WO2015134808A2 - Probiotic formulations and methods for use - Google Patents

Probiotic formulations and methods for use Download PDF

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Publication number
WO2015134808A2
WO2015134808A2 PCT/US2015/019059 US2015019059W WO2015134808A2 WO 2015134808 A2 WO2015134808 A2 WO 2015134808A2 US 2015019059 W US2015019059 W US 2015019059W WO 2015134808 A2 WO2015134808 A2 WO 2015134808A2
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Prior art keywords
poly
composition
reuteri
biofilm
microsphere
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PCT/US2015/019059
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English (en)
French (fr)
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WO2015134808A3 (en
Inventor
Michael Bailey
Steven J. Goodman
Lauren O. Bakaletz
Gail BESNER
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Research Institute At Nationwide Children's Hospital
Ohio State Innovation Foundation
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Priority to JP2016555766A priority Critical patent/JP6754694B2/ja
Priority to AU2015227075A priority patent/AU2015227075A1/en
Priority to EP19154908.8A priority patent/EP3536168A3/de
Priority to EP15710693.1A priority patent/EP3113630B1/de
Priority to CA2941694A priority patent/CA2941694A1/en
Application filed by Research Institute At Nationwide Children's Hospital, Ohio State Innovation Foundation filed Critical Research Institute At Nationwide Children's Hospital
Publication of WO2015134808A2 publication Critical patent/WO2015134808A2/en
Publication of WO2015134808A3 publication Critical patent/WO2015134808A3/en
Priority to US15/257,673 priority patent/US10369176B2/en
Priority to US15/649,352 priority patent/US10624934B2/en
Priority to AU2019201644A priority patent/AU2019201644A1/en
Priority to US16/449,320 priority patent/US11452748B2/en
Priority to US16/820,463 priority patent/US11497780B2/en
Priority to AU2021203170A priority patent/AU2021203170B2/en
Priority to US17/885,446 priority patent/US20230285475A1/en
Priority to US17/955,413 priority patent/US20240075079A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/195—Proteins from microorganisms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1611—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
    • A61K9/1647—Polyesters, e.g. poly(lactide-co-glycolide)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1658—Proteins, e.g. albumin, gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
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    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
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    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/32—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the digestive tract
    • A23V2200/3202—Prebiotics, ingredients fermented in the gastrointestinal tract by beneficial microflora
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/32—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the digestive tract
    • A23V2200/3204—Probiotics, living bacteria to be ingested for action in the digestive tract

Definitions

  • This disclosure relates to novel probiotic formulations and methods for using same for treating or preventing disease.
  • EHEC Enterohemorrhagic Escherichia coli
  • EPEC enteropathogenic E. coli
  • Citrobacter rodentium is a murine pathogen that is widely used to model human EPEC and EHEC infection, because mice are relatively resistant to both EPEC and EHEC.
  • C. rodentium results in colonic pathology that is nearly indistinguishable from that produced by EPEC and EHEC in humans (Borenshtein, M. et al. (2008) Utility of the Citrobacter rodentium Infection Model in Laboratory Mice, Curr Opin Gastroenterol, 24:32-7; Luperchio and Schauer, (2001) Molecular Pathogenesis of Citrobacter rodentium and Transmissible Murine Colonic Hyperplasia, Microbes Infect, 3:333-40; Mundy, T.
  • C. rodentium possesses a homologue of the locus of enterocyte effacement (LEE) pathogenicity island carried by EPEC and EHEC that encodes for the effector proteins necessary for the development of attaching and effacing (A/E) lesions.
  • LEE enterocyte effacement
  • A/E attaching and effacing
  • lesions are accompanied by the development of colonic hyperplasia, and pathological colitis marked by epithelial defects and leukocyte infiltration (Luperchio and Schauer (2001) Molecular Pathogenesis of Citrobacter rodentium and Transmissible Murine Colonic Hyperplasia, Microbes Infect. 3:333-40).
  • enteric pathogens In order to cause disease, enteric pathogens must either adhere to or penetrate/invade host epithelial cells. Thus, interaction with epithelial cells is the first step in pathogenicity for all enteric pathogens, and this step can be studied through the use of A/E pathogens by assessing colonic colonization and resultant pathology.
  • Colonization of A/E pathogens in the colon is dependent upon the composition of the intestinal microbiota. Inducing dysbiosis (the disruption of the native populations of beneficial bacteria) within the colonic microbiota by administering antibiotics (Wlodarska, B. et al, (2011) Antibiotic Treatment Alters the Colonic Mucus Layer and Predisposes the Host to Exacerbated Citrobacter rodentium-lnduced Colitis, Infect Immun, 79: 1536-45) or by inducing an inflammatory response (Lupp, M. L.
  • Colonic dysbiosis can further exacerbate the inflammatory response to the colonic pathogen (Wlodarska, B. et al., (2011) Antibiotic Treatment Alters the Colonic Mucus Layer and Predisposes the Host to Exacerbated Citrobacter rodentium-lnduced Colitis, Infect Immun.
  • dysbiosis can propagate inflammatory responses in genetically susceptible individuals, as evidenced by the findings of dysbiosis in patients with inflammatory bowel disease (Machiels, et al., (2013) A Decrease of the Butyrate-Producing Species Roseburia hominis and Faecalibacterium prausnitzii Defines Dysbiosis in Patients with Ulcerative Colitis, Gut, published online first September 10, 2013; Morgan, et al., (2012) Dysfunction of the Intestinal Microbiome in Inflammatory Bowel Disease and Treatment, Genome Biol., 13:R79) or irritable bowel syndrome (Carroll, et al., (2012) Alterations in Composition and Diversity of the Intestinal Microbiota in Patients with Diarrhea-Predominant Irritable Bowel Syndrome, Neurogastroenterol Motil.
  • Probiotics are live microbes that when ingested in high enough quantities confer a health benefit for the host (Food and Agriculture Organization of the United Nations and World Health Organization, "Health and Nutritional Properties of Probiotics in Food
  • probiotic microbes have the capacity to enhance immune system activity, but fewer probiotic microbes have anti-inflammatory effects.
  • Lactobacillus reuteri is a commonly used probiotic that has been shown to regulate the mammalian and avian intestinal immune system (Lin, et al, (2008) Probiotic Lactobacillus reuteri Suppress Proinflammatory Cytokines Via C-Jun, Inflamm Bowel Dis., 14: 1068-83). Studies in vitro demonstrate that some strains of L.
  • reuteri can suppress the ability of myeloid cells to produce inflammatory cytokines (such as TNF-a) through a down-regulation of cell signal transduction pathways ⁇ e.g., c-Jun-dependent activator protein 1 (AP-1)) (Jones and
  • L. reuteri Other strains of L. reuteri, such as ATCC23272, can down-regulate both cytokine and chemokine production by colonic epithelial cells stimulated with C. rodentium. L.
  • Lactobacillus reuteri can also reduce colonic inflammation in both juvenile and adult animals (Eaton, A. et al., (2011) Probiotic Lactobacillus reuteri Ameliorates Disease Due to Enterohemorrhagic Escherichia coli in Germfree Mice, Infect Immun., 79: 185-91; Schreiber, et al, (2009) Lactobacillus reuteri Prevents Colitis by Reducing P-Selectin- Associated Leukocyte- and Platelet-Endothelial Cell Interactions, Am J Physiol Gastrointest Liver Physiol, 296:G534- 42).
  • L. reuteri was able to prevent pathogen translocation and the development of systemic inflammatory responses, but it was not able to reduce all aspects of colonic pathology (Mackos, et al., (2013) Probiotic Lactobacillus reuteri Attenuates the Stressor-Enhanced Severity of Citrobacter rodentium Infection, Infect Immun, 81 :3253-63). [0013] Moreover, the effects of L. reuteri on the host were short-lived and no longer evident after daily administration was terminated. These studies demonstrate the
  • probiotics can effectively prevent pathogen colonization due to either direct (e.g., production of antimicrobial defenses) or indirect (e.g., stimulation of host defenses) mechanisms. Few probiotic species are able to both prevent pathogen colonization and limit excessive inflammatory responses. This is important, however, because excessive colonic inflammation in response to colonic infection can lead to the development of protracted illness, such as post-infectious irritable bowel syndrome. Thus, the development of probiotics that are able to prevent excessive immune responses to colonic pathogens, while still maintaining anti-bacterial immunity would have the ability to prevent both short-term and longer-term health effects of enteric infection.
  • probiotic bacteria with prebiotic substances to help stimulate the exclusive growth of the probiotic species and provide the bacteria in the form of a biofilm on a biocompatible microsphere, which has greater efficacy and duration. It has been shown that probiotic biofilms can be grown on surfaces as a means to introduce bacteria into the site of wounds, where a formulation comprising a plaster or dressing based on a hydrocolloid that is a natural gelatin to treat wounds (i.e., EP2450062). However, there is an unmet need for fewer probiotic doses and greater efficacy of probiotic bacteria and its appropriate formulation in the methods as disclosed herein, to the best of Applicants' knowledge, has not yet heretofore been disclosed.
  • This technology provides methods of formulation, which enhance the efficiency and durability of introducing probiotic strains at a site. It specifically bypasses the rate limiting step of bio film formation.
  • This technology is useful for gastrointestinal gut health and any aspects where probiotic bacteria need to establish, e.g., the gastrointestinal tract, wound healing, skin, vaginal, oral, water purification.
  • Probiotics are a natural way to protect and restore gut microbiota to a healthy state.
  • probiotic bacteria as typically delivered
  • One of the rate limiting steps is the capacity of introduced bacteria to form a lasting biofilm.
  • bacteria are already in the form of a biofilm (a surface adhered community) as opposed to planktonic (free-living), they more readily establish and persist.
  • the positive effects of probiotic bacteria can be enhanced by providing them in a biofilm state; this can readily be accomplished by growing the bacteria on the surface of a biocompatible and nontoxic microsphere.
  • Biocompatible microspheres can be biodegradable polymers, nonbiodegradable polymers, a metal, or a combination thereof. When this surface is in the form of a microsphere, prebiotic and/or prebiofilmic substances can be added as cargo to facilitate establishment and maintenance of the probiotic bacterial biofilm.
  • Microspheres have added value in ideally providing diffusible prebiotic (nutritional supplementation specific/exclusive to probiotic bacteria) cargo that can help promote probiotic bacterial establishment and survival while limiting pathogenic bacterial challenge.
  • prebiotic nutritional supplementation specific/exclusive to probiotic bacteria
  • the biofilm state is advantageous in establishing in the murine gut over the same bacteria in planktonic form.
  • Citrobacter rodentium than L. reuteri in its planktonic form. Based on these results, highly integrated examples are developed that yield novel formulations of probiotics that provide greater and more lasting effects against dysbiosis preventing or even treating gut
  • the bio film-generating probiotic bacterium adheres to the surface of the biocompatible microsphere and generates a biofilm.
  • the biocompatible microsphere has either a solid or hollow core. When the biocompatible microsphere has a hollow core, it can carry a prebiotic and any nutritional supplementation for the probiotic bacterium as a cargo. The prebiotic can be encapsulated within the hollow core.
  • the microsphere can also carry a drug, or a compound, or an agent, which is selective against a pathogen, that in one aspect, maycompete with the health-inducing bacterium in the composition.
  • a novel probiotic formulation can also contain a prebiofilmic, which is a substance that supports biofilm formation and/or durability, and in one aspect, the prebiofilmic is a DNA binding polypeptide or protein and/or a DNABII polypeptide or protein or a fragment thereof that supports biofilm formation and/or durability.
  • composition comprising, or alternatively consisting essentially of, or yet further consisting of, a biocompatible microsphere, a biofilm-generating probiotic bacterium and a prebiotic, wherein the prebiotic comprises, or alternatively consisting essentially of, or yet consisting of, a nutritional supplementation for the probiotic bacterium.
  • the composition further comprises, or alternatively consists essentially of, or yet further consisting of, a carrier, such as a pharmaceutically acceptable carrier or a biocompatible scaffold.
  • the composition is formulated in a dosage form.
  • Suitable dosage forms include, but are not limited to a suppository, a powder, a liquid, a capsule, a chewable tablet, a swallowable tablet, a buccal tablet, a troche, a lozenge, a soft chew, a solution, a suspension, a spray, a tincture, a decoction, an infusion, and combinations thereof.
  • This disclosure also provides a method for preparing the above-noted composition, the method comprising, or alternatively consisting essentially of, or yet further consisting of, admixing a biocompatible microsphere with a biofilm-generating probiotic bacterium, a prebiotic, and in one aspect, further admixing a prebiofilmic.
  • the method further comprises, or alternatively consists essentially of, or yet further consists of, admixing an effective amount of a nutritional supplement for the probiotic bacterium.
  • composition comprising, or alternatively consisting essentially of, or yet further consisting of, a PGLA-biocompatible microsphere, one or more biofilm-generating probiotic bacterium comprising at least Lactobacillus reuteri ("L.
  • composition may further comprise, or alternatively consist essentially of, or yet further consist of, an effective amount of an HU polypeptide or protein.
  • composition can further comprise a pharmaceutically acceptable carrier or a biocompatible scaffold and is optionally formulated as a suppository.
  • This disclosure also provides a method for treating or preventing a disease suitable treated by a bio film in a subject, such as inflammatory bowled disease, necrotizing enterocolitis (NEC) or psychological disorders or mood disorders, in a subject comprising, or alternatively consisting essentially of, or yet further consisting of, administering to a subject in need thereof, an effective amount of a composition comprising, or alternatively consisting essentially of, or yet further consisting of, a composition as described herein.
  • a disease suitable treated by a bio film in a subject such as inflammatory bowled disease, necrotizing enterocolitis (NEC) or psychological disorders or mood disorders
  • kits comprising, or alternatively consisting essentially of, or yet consisting of, a composition as described herein and instructions for use diagnostically or therapeutically.
  • FIGS. 1 A and IB illustrate that L. reuteri biofilm structural integrity relies on the presence of DNABII family proteins. Confocal microscopy images of in vitro L. reuteri biofilms stained with LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probes). L. reuteri biofilms were grown for 24 hours at 37°C and 5% C02, at which time they were treated with a 1 :50 dilution of either A) rabbit naive serum, B) rabbit anti-integration host factor polypeptide ("IHF”) , or media with nothing added (data not shown) for 16 hours. Anti-IHF treatments resulted in a 20% decrease in maximum height, 35% decrease in average thickness, and 41% decrease in biomass (data not shown).
  • IHF rabbit anti-integration host factor polypeptide
  • FIG. 2 illustrates that Prebiotic compounds increase probiotic biofilms in average thickness and biomass.
  • Addition of 10 ⁇ S. mutans HU to L. reuteri biofilm at time of seeding increased average thickness and biomass 33%, and 55%, respectively.
  • Addition of 10 ⁇ calf thymus DNA increased average thickness 44% and biomass 68%.
  • FIG. 3 illustrates that L. reuteri in vivo colonization and retention with a single oral administration.
  • FIG. 4 illustrates that L. reuteri biofilm grown with PLGA microspheres and HU reduces C. rodentium spleen colonization more effectively than biofilm and planktonic L. reuteri.
  • FIG. 5 shows the results of a study establishing that compositions of this disclosure reduce inflammation and antagonize bacterial pathogens in an animal model of NEC.
  • SEQ ID NO: 1 Full Length Wild type (wt) 86-028NP Haemophilus influenzae IhfA; Genbank accession No.: AAX88425.1, last accessed March 21, 2011 :
  • SEQ ID NO: 2 Full Length wild-type 86-028NP Haemophilus influenzae HU, Genbank accession No.: YP_248142.1, last accessed March 21, 2011 :
  • SEQ ID NO: 3 Full Length wt R2846 Haemophilus influenzae IhfA, Genbank accession No.: AD096375, last accessed March 21, 2011 :
  • SEQ ID NO: 4 Full Length wild-type Rd Haemophilus influenzae IhfA; Genbank accession No.: AAC22959.1, last accessed March 21, 2011 :
  • SEQ ID NO: 5 Full Length wild-type E. coli K12 IhfA; Genbank accession No.: AAC74782.1, last accessed March 21 , 2011 :
  • SEQ ID NO: 6 Full Length wild-type P. aeruginosa PA 01 IhfA; Genbank accession No.: AAG06126.1, last accessed March 21, 2011 : MGALTKAEIAERLYEELGLNKREA KELVELFFEEIRQALEHNEQVKLSGFGNFDLRDKRQRPGRNPKTGEEIPITARRVVTF RPGQKLKARVEAYAGTKS
  • SEQ ID NO: 13 ⁇ -3 and a-3 TFRPGQ and KLKARVEAYAGTKS
  • SEQ ID NO: 14 E. coli hup A, Genbank accession No. : AP 003818, Last accessed March 21, 2011 :
  • SEQ ID NO: 15 E. coli hupB, Genbank accession No.: AP 001090.1, Last accessed March 21, 2011 :
  • SEQ ID NO: 20 DNABII binding consensus sequence: WATCAANNNNTTR wherein W is A or T, N is any base and R is a purine.
  • SEQ ID NO: 22 E. coli IHFbeta: GRNPKTGDKVEL
  • SEQ ID NO: 23 E. coli HUalpha: GRNPQTGKEIKI
  • SEQ ID NO: 24 E. coli HUbeta: GRNPQTGKEITI
  • a bacterium includes a plurality of bacteria, including mixtures thereof.
  • compositions and methods include the recited elements, but do not exclude others.
  • Consisting essentially of when used to define compositions and methods shall mean excluding other elements of any essential significance to the combination for the intended use.
  • a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives and the like.
  • Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention. Embodiments defined by each of these transition terms are within the scope of this invention.
  • a "biofilm” intends a thin layer or an organized community of microorganisms that at times can adhere to the surface of a structure, that may be organic or inorganic, together with the polymers, such as DNA, that they secrete and/or release.
  • the biofilms are very resistant to microbiotics and antimicrobial agents. They live on gingival tissues, teeth, and restorations, causing caries and periodontal disease, also known as periodontal plaque disease. They also cause chronic middle ear infections. Biofilms can also form on the surface of dental implants, stents, catheter lines and contact lenses. They grow on
  • a "prebiotic” intends a nutritional supplement for the probiotic bacterium.
  • Prebiotics are food ingredients, for example, oligosaccharides, that are non-digestible by a subject (e.g., by a mammal such as a human), and that stimulates the growth or activity of one or more beneficial bacteria and/or inhibit the growth or activity of one or more pathogenic bacteria.
  • a prebiotic may selectively stimulate the growth and/or activity of one or a limited number of bacteria in the subject.
  • a "prebiofilmic” intends a substance that supports biofilm formation and durability, for example the prebiofilmic can be a substance that supports the extracellular matrix of the biofilm like an eDNA binding polypeptide or protein or alternatively a substrate that can be converted into a substance that facilitate adhesion, e.g. sucrose.
  • a "DNABII polypeptide or protein” intends a DNA binding protein or polypeptide that is composed of DNA-binding domains and thus have a specific or general affinity for DNA. In one aspect, they bind DNA in the minor grove.
  • Non- limiting examples of DNABII proteins are an integration host factor (IHF) protein and a histone-like protein from E. coli strain U93 (HU), examples of which are provided in SEQ ID NOs: 1 to 24 and additional strains and polypeptides are provided in Table 4. Also intended are polypeptide fragments and equivalent polypeptides that have amino acid modifications that do not substantially change the biological activity of the protein or polypeptides, or active fragment thereof.
  • Active fragments thereof include, for example, the c-terminal half or c-terminal third of the protein or polypeptide.
  • Other DNA binding proteins that can be associated with the biofilm include DPS (Genbank Accession No.: CAA49169), H-NS (Genbank Accession No.:
  • CAA47740 Hfq (Genbank Accession No.: ACE63256), CbpA (Genbank Accession No.: BAA03950) and CbpB (Genbank Accession No.: NP 418813), as well as equivalent polpyeptides and active fragments thereof.
  • a "microsphere” intends a biofilm-carrying and/or compound-carrying (e.g., drug- carrying) particulate or granular material within the particular size range recited.
  • a microsphere consisting of particles 50 millimeters or less in diameter, and 1 micron or more (e.g., 1 to 100 or alternatively, or alternatively, 1 to 75 microns, or alternatively 1 to 50, or alternatively 1 to 25, or alternatively 1 to 10 microns) in diameter.
  • Non-limiting examples of such include hollow microspheres that can, in some aspects, contain a pharmaceutical or drug, microcapsules (in which the excipient forms a skin or shell that surrounds and contains a cargo, such as a drug), and microparticles, which are used as a generic term for any particles in the recited size range, whether spherical or not, as those terms are typically used in the art.
  • a “biodegradable polymer” intends polymers that are biocompatible and can degrade in vivo by bodily processes to products that are readily disposable by the body and should not accumulate in the body.
  • biocompatible it is meant that the components of the delivery system will not cause tissue injury or injury to the human biological system.
  • polymers and excipients that have had history of safe use in humans or with GRAS
  • biocompatibility it is meant that the ingredients and excipients used in the composition will ultimately be “bioabsorbed” or cleared by the body with no adverse effects to the body.
  • bioabsorbable refers to microspheres made from materials which undergo bioabsorption in vivo over a period of time such that long term accumulation of the material in the patient is avoided.
  • the biocompatible nanoparticle is bioabsorbed over a period of less than 2 years, preferably less than 1 year and even more preferably less than 6 months. The rate of bioabsorption is related to the size of the particle, the material used, and other factors well recognized by the skilled artisan.
  • a mixture of bioabsorbable, biocompatible materials can be used to form the microspheres used in this invention.
  • IHF protein is a bacterial protein that is used by bacteriophages to incorporate their DNA into the host bacteria. These are DNA binding proteins that function in genetic recombination as well as in transcription and translational regulation. They also bind extracellular microbial DNA. The genes that encode the IHF protein subunits in E. coli are himA (Genbank accession No.: POA6X7.1) and himD
  • HU or "histone-like protein from E. coli strain U93” refers to a class of heterodimeric proteins typically associated with E. coli. HU proteins are known to bind DNA junctions. Related proteins have been isolated from other microorganisms. The complete amino acid sequence of E. coli HU was reported by Laine et al. (1980) Eur. J. Biochem. 103(3):447-481. Antibodies to the HU protein are commercially available from Abeam. Non-limiting examples of such are provided in the attached sequence listing.
  • protein protein
  • peptide and “polypeptide” are used interchangeably and in their broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics.
  • the subunits may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g., ester, ether, etc.
  • a protein or peptide must contain at least two amino acids and no limitation is placed on the maximum number of amino acids which may comprise a protein's or peptide's sequence.
  • amino acid refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics.
  • a "c-terminal polypeptide” intends the c-terminal half or c-terminal third of a polypeptide.
  • the c-terminal polypeptide would comprise amino acids 46 through 90 or amino acids 60 through 90.
  • the term intends the c-terminal 20 amino acids from the carboxy terminus.
  • a "n-terminal polypeptide” intends the n-terminal half of a polypeptide.
  • the c-terminal polypeptide would comprise amino acids 1 through 45.
  • the term intends the c-terminal 20 amino acids from the amino terminus.
  • polynucleotide and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown.
  • polynucleotides a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger R A (mRNA), transfer R A, ribosomal R A, RNAi, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers.
  • a polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide.
  • the sequence of nucleotides can be interrupted by non-nucleotide components.
  • a polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.
  • the term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of this invention that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
  • a polynucleotide is composed of a specific sequence of four nucleotide bases:
  • polynucleotide sequence is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.
  • isolated or recombinant refers to molecules separated from other DNAs or RNAs, respectively that are present in the natural source of the macromolecule as well as polypeptides.
  • isolated or recombinant nucleic acid is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.
  • isolated is also used herein to refer to polynucleotides, polypeptides, antibodies and proteins that are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides.
  • the term "isolated or recombinant” means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, which are normally associated in nature.
  • an isolated cell is a cell that is separated from tissue or cells of dissimilar phenotype or genotype.
  • An isolated polynucleotide is separated from the 3 ' and 5 ' contiguous nucleotides with which it is normally associated in its native or natural environment, e.g., on the chromosome.
  • a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof does not require "isolation" to distinguish it from its naturally occurring counterpart.
  • nucleic acid, polynucleotide, polypeptide or protein mentioned herein also includes equivalents thereof.
  • an equivalent intends at least about 70%, or alternatively 80 % homology or identity and alternatively, at least about 85 %, or
  • a polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) having a certain percentage (for example, 80%, 85%, 90%, or 95%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences.
  • the alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Current Protocols in Molecular Biology (Ausubel et al, eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1.
  • default parameters are used for alignment.
  • a preferred alignment program is BLAST, using default parameters.
  • Homology refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or “non-homologous" sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present invention.
  • expression refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in an eukaryotic cell.
  • encode refers to a polynucleotide which is said to "encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for the polypeptide and/or a fragment thereof.
  • the antisense strand is the
  • a "subject" or “patient” of diagnosis or treatment is a cell or an animal such as a mammal or a human.
  • Non-human animals subject to diagnosis or treatment and are those subject to infections or animal models, for example, simians, murines, such as, rats, mice, chinchilla, canine, such as dogs, leporids, such as rabbits, livestock, sport animals and pets.
  • the terms “treating,” “treatment” and the like are used herein to mean obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disorder or sign or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disorder and/or adverse effect attributable to the disorder.
  • To "prevent” intends to prevent a disorder or effect in vitro or in vivo in a system or subject that is predisposed to the disorder or effect.
  • An example of such is preventing the formation of a biofilm in a system that is infected with a microorganism known to produce one.
  • the term “culturing” refers to the in vitro propagation of cells or organisms on or in media of various kinds. It is understood that the descendants of a cell grown in culture may not be completely identical (i.e., morphologically, genetically, or phenotypically) to the parent cell. By “expanded” is meant any proliferation or division of cells.
  • “Pharmaceutically acceptable carriers” refers to any diluents, excipients or carriers that may be used in the compositions of the invention.
  • Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium
  • Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. They are preferably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like and consistent with conventional pharmaceutical practices.
  • a “biocompatible scaffold” refers to a scaffold or matrix for with the ability to support biofilm proliferation upon administration to a subject.
  • a biocompatible scaffold is a precursor to an implantable device which has the ability to perform its intended function, with the desired degree of incorporation in the host, without eliciting an undesirable local or systemic effects in the host.
  • Biocompatible scaffolds are described in U.S. Patent Nos. 6,638,369 and 8,815,276; [0089] "Administration" can be effected in one dose, continuously or intermittently throughout the course of treatment.
  • Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents are known in the art. Route of administration can also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated and target cell or tissue. Non-limiting examples of route of administration include oral administration, vaginal, nasal administration, injection, topical application and by
  • the term "effective amount" refers to a quantity sufficient to achieve a beneficial or desired result or effect.
  • the effective amount will depend on the type and severity of the condition at issue and the characteristics of the individual subject, such as general health, age, sex, body weight, and tolerance to pharmaceutical compositions.
  • the effective amount is the amount sufficient to result in a protective response against a pathogen.
  • the effective amount is the amount sufficient to result in antibody generation against the antigen.
  • the effective amount is the amount required to confer passive immunity on a subject in need thereof.
  • the amount is sufficient to accomplish one or more of 1) clear pathogen; 2) restore healthy mircobiota; 3) modulate the immune system; and 4) maintain metabolism and metabolic pathways.
  • the effective amount will depend on the size and nature of the application in question. It will also depend on the nature and sensitivity of the in vitro target and the methods in use. The skilled artisan will be able to determine the effective amount based on these and other considerations.
  • the effective amount may comprise one or more administrations of a composition depending on the embodiment.
  • the agents and compositions can be used in the manufacture of medicaments and for the treatment of humans and other animals by administration in accordance with conventional procedures, such as an active ingredient in pharmaceutical compositions.
  • An agent of the present invention can be administered for therapy by any suitable route of administration. It will also be appreciated that the preferred route will vary with the condition and age of the recipient and the disease being treated.
  • Necrotizing enterocolitis is a medical condition primarily seen in premature infants where portions of the bowel undergo necrosis (tissue death). It occurs postnatally (i.e., is not seen in stillborn infants) and is the second most common cause of mortality. 7% of all neonatal intensive care unit admissions are NEC related. The mortality rate is 12%
  • Diarrheal illness occurs in approximately four billion individuals per year and causes more than two million deaths worldwide.
  • A/E pathogens attaching and effacing (A/E) pathogens, which upon colonization induce diarrheal disease that is associated with an increase in inflammatory cytokines and structural changes to colonic tissue.
  • This acute infection can have a lasting effect on gut health, and infection with A/E pathogens and excessive inflammatory responses are known risk factors for the development of postinfectious irritable bowel syndrome.
  • Probiotics are a natural way to protect and restore gut microbiota to a healthy state and have been shown to promote health distal to the site of colonization. See Mackos et al. (2013) Infection and Immunity 81, No. 9 (3253-3262). Unfortunately, even under optimal conditions, probiotic bacteria fail to establish, or sufficiently persist, minimizing the magnitude and duration of their healthful effects.
  • One of the rate limiting steps is the capacity of introduced bacteria to form a lasting biofilm. When bacteria are already in the form of a biofilm (a surface adhered community) as opposed to planktonic (free-living), they more readily establish and persist.
  • Biocompatible microspheres can be biodegradable polymers, non-biodegradable polymers, a metal, or a combination thereof.
  • prebiotic and/or prebiofilmic substances can be added as cargo to facilitate establishment and maintenance of the probiotic bacterial biofilm.
  • Microspheres have added value in ideally providing diffusible prebiotic (nutritional supplementation specific/exclusive to probiotic bacteria) cargo that can help promote probiotic bacterial establishment and survival while limiting pathogenic bacterial challenge.
  • prebiotic nutritional supplementation specific/exclusive to probiotic bacteria
  • the biofilm state is advantageous in establishing in the murine gut over the same bacteria in planktonic form.
  • Citrobacter rodentium than L. reuteri in its planktonic form. Based on these results, three highly integrated examples are developed that yield novel formulations of probiotics that provide greater and more lasting effects against dysbiosis preventing or even treating gut pathogenesis with a far reduced need for patient compliance.
  • the biofilm-generating probiotic bacterium adheres to the surface of the biocompatible microsphere and generates a biofilm.
  • the biocompatible microsphere has either a solid or hollow core.
  • the biocompatible microsphere can carry a prebiotic and any nutritional supplementation for the probiotic bacterium as a cargo.
  • the prebiotic can be encapsulated within the hollow core.
  • the microsphere can also carry a drug, or a compound, or an agent, which is selective against the growth or proliferation of a pathogen.
  • a novel probiotic formulation may also contain a prebiofilmic, which a substance that supports biofilm formation and durability, specifically, the prebiofilmic is a DNA binding polypeptide or protein and/or a DNABII polypeptide or protein, a fragment and/or an equivalent of each thereof.
  • a prebiofilmic which a substance that supports biofilm formation and durability
  • the prebiofilmic is a DNA binding polypeptide or protein and/or a DNABII polypeptide or protein, a fragment and/or an equivalent of each thereof.
  • One or more drug, compound or agent as well as one or more prebiofilmic can be within a single microsphere.
  • the prebiotic can support the growth of any probiotic bacteria, including biofilm- generating bacteria.
  • the prebiotic is usually one or more of a water-soluble carbohydrate, such as inulin, oligofructose, fructo-oligosaccharide, galacto-oligosaccharide, glucose, maltose, maltodextrins, polydextrose, sucrose, fructose, lactose, isomaltulose, polyols, and glycerol.
  • a water-soluble carbohydrate such as inulin, oligofructose, fructo-oligosaccharide, galacto-oligosaccharide, glucose, maltose, maltodextrins, polydextrose, sucrose, fructose, lactose, isomaltulose, polyols, and glycerol.
  • the combination of various prebiotics can be used to support the growth of probiotics.
  • Probiotics are any type of micro-organisms that have health benefits. Probiotics are also commonly consumed as part of fermented foods with specially added active live cultures, such as in yogurt, soy yogurt, or as dietary supplements. Probiotics can also be taken as a suppository. Some limiting examples of probiotics are L. acidophilus, L. crispatus, L. gasseri, group L. delbrueckii, L. salivarius, L. casei, L. paracasei, L. plantarum, L.
  • rhamnosus L. reuteri, L. brevis, L. buchneri, L.fermentum, L. rhamnosus, B. adolescentis, B. angulation, B. bifidum, B. breve, B. catenulatum, B. infantis, B. lactis, B. longum, B.
  • Probiotics support anti-bacterial immunity by preventing pathogen colonization and/or limiting excessive inflammatory responses. Without being bound by theory, the probiotics down-regulate cytokine and chemokine production.
  • the biocompatible microsphere can be one or more of a biodegradable polymer, a non-biodegradable polymer, a metal, or a mixture thereof.
  • the biodegradable polymer can be selected from, but not limited to, poly(lactic-co-glycolic acid) or PLGA; polycaprolactone or PLC; Chitosan; Gelatin; DNA hydrogen; acetalated dextran; poly(lactide); poly(glycolide); poly(lactide-co-glycolide); poly(lactic acid); poly(glycolic acid); poly(lactic acid-co-glycolic acid); poly(lactide)/poly(ethylene glycol) copolymers; poly(glycolide)/poly(ethylene glycol) copolymer; poly(lactide-co-glycolide)/poly(ethylene glycol) copolymers; poly(lactic acid)/poly(ethylene glycol) copolymer; poly(glycolic acid)/poly(ethylene glyco
  • the non-biodegradable polymer can be selected from, but not limited to, poly(ethylene vinyl acetate), poly( vinyl acetate), silicone polymers, polyurethanes, polysaccharides such as a cellulosic polymers and cellulose derivatives, acyl substituted cellulose acetates and derivatives thereof, copolymers of poly(ethylene glycol) and poly(butylene terephthalate), polystyrenes, polyvinyl chloride, polyvinyl fluoride, poly( vinyl imidazole), chorosulphonated polyolefms, polyethylene oxide, and copolymers and blends thereof.
  • the metal can be selected from, but not limited to, cobalt, chromium, gold, nickel, platinum, stainless steel, titanium,
  • the microspheres are selected to facilitate the endurance and robustness of the probiotic biofilms are identified and characterized. It has been shown that probiotic biofilms formed on the biodegradable (and FDA approved) surface, poly(lactic-co-glycolic acid) (PLGA) yields biofilms that are superior at preventing pathogen translocation through the epithelial barrier. Other FDA approved or generally regarded as safe (GRAS) materials that can be used to create surfaces to grow biofilms are also examined. The results using biological effectiveness and durability in animal models and shelf life as the base criteria are prioritized. Finally, to further improve the effectiveness of the introduction and maintenance of the probiotic biofilm, prebiotic substances to the probiotic biofilm surface by way of diffusible cargo within the microspheres are provided.
  • PLGA poly(lactic-co-glycolic acid)
  • composition comprising, or alternatively consisting essentially of, or yet further consisting of, a biocompatible microsphere, a biofilm-generating probiotic bacterium and a prebiotic, wherein the prebiotic comprises, or alternatively consists essentially of, or yet further consists of a nutritional food source or supplement for the culturing and/or growth of the probiotic bacterium.
  • the composition can further comprise a prebiofilmic.
  • the prebiofilmic comprises a substance that supports biofilm formation and durability, specifically; the prebiofilmic can be a DNA binding polypeptide or protein and/or a DNABII polypeptide or protein.
  • the composition is frozen, for example flash frozen.
  • the composition is lyophilized or dried in powder form.
  • composition for administration as a suppository or in ingestible form (e.g., tablet).
  • the composition can further comprise a mixture of the above -noted microspheres, e.g., a mixture containing two or more probiotic bacterium and/or two or prebiofilmics and/or two or more nutritional and/or supplement to support the culturing and/or growth of the probiotic bacterium.
  • the prebiotic comprises a water-soluble carbohydrate selected from, but not limited to, one or more of inulin, oligofructose, fructo-oligosaccharide, galacto-oligosaccharide, glucose, maltose, maltodextrins, polydextrose, sucrose, fructose, lactose, isomaltulose, polyols, glycerol, and combinations thereof.
  • the composition further comprises a solid or a liquid carrier, such as a pharmaceutically acceptable carrier.
  • the prebiotic and prebiofilmic are selected in each composition to specifically support the growth of the probiotic bacterium.
  • the composition comprises an effective amount of sucrose, glycerol and optionally HU polypeptide or protein, to support the growth and maintenance of the probiotic when administered to the subject or patient.
  • Non-limiting examples of prebioflimic compositions include, without limitation, one or more of the polypeptides provided in SEQ ID NOs: 1 to 24, a c-terminal fragment thereof, or a n- terminal fragment thereof, or the additional strains and polypeptides and fragments thereof, such as the full length or the c-terminal fragment or the n-terminal fragment of those provided in Table 4, and equivalents of each thereof. Additional nutritional supplements for the support of other probiotic bacterium are disclosed in Bergey's Manual of Determinative Bacteriology, 9 th Ed, Ed. Holt et al, Williams Wilkins (1994),
  • Non-limiting examples of a probiotic bacterium for use in the composition includes, without limitation, one or more of L. acidophilus, L. crispatus, L. gasseri, group L.
  • delbrueckii L. salivarius, L. casei, L. paracasei, L. plantarum, L. rhamnosus, L. reuteri, L. brevis, L. buchneri, L. fermentum, L. rhamnosus, B. adolescentis, B. angulation, B. bifidum, B. breve, B. catenulatum, B. infantis, B. lactis, B. longum, B. pseudocatenulatum, S.
  • thermophiles or a combination thereof.
  • one or more bacterium can be combined in a single composition.
  • the probiotic bacterium is Lactobacillus reuteri.
  • the bacteria are available from commercial sources, such as the American Type Culture Collection (ATCC).
  • ATCC American Type Culture Collection
  • the one or more probiotic bacterium in the composition supports anti-bacterial immunity.
  • the one or more probiotic bacterium in the composition prevents pathogen colonization and/or limits excessive inflammatory responses by down-regulating cytokine and chemokine production.
  • the composition further comprises an agent, and the agent is selective against a pathogen, such as a competing pathogen.
  • the biocompatible microsphere comprises one or more of a biodegradable polymer, a non-biodegradable polymer, a metal, or a combination thereof.
  • the microsphere comprises a solid core.
  • the microsphere comprises a hollow core.
  • the prebiotic is encapsulated within the hollow core of the microsphere.
  • the disclosure provides a composition comprising, or alternatively consisting essentially of, or yet further consisting of, a PGLA-biocompatible microsphere, one or more biofilm-generating probiotic bacterium, and a nutritional supplementation comprising one or more of sucrose or glycerol in an amount to support the growth of the probiotic bacterium.
  • the biofilm-generating probiotic bacterium may comprise Lactobacillus reuteri ("Z. reuteri").
  • the composition may further comprise, or alternatively consist essentially of, or yet further consist of, an effective amount of HU polypeptide or protein.
  • the composition can further comprise a pharmaceutically acceptable carrier or a
  • biocompatible scaffold and is optionally formulated as a suppository.
  • the size of the microsphere can range from about 0.5 microns to about 100 microns. In certain embodiments, the microsphere is less than about 100 microns in diameter. In other embodiments, the microsphere is less than about 50 microns, or less than about 40 microns, or less than about 30 microns, less than about 20 microns, less than about 10 microns, or less than about 5 microns, or less than 3 microns to 0.5 microns in diameter.
  • the microsphere is from about 0.5 microns to about 90 microns, or to about 80 microns, or to about 70 microns, or to about 60 microns, or to about 50 microns, or to about 40 microns, or to about 30 microns, or to about 20 microns, or about 10 microns, or about 5 microns, or about 3 microns, or about 2 microns, or about 1 micron, in diameter.
  • the diameter is from about 1 to about 100, or alternatively from about 1 to about 75, or alternatively from about 1 to about 50, or alternatively from about 1 to about 25, or alternatively from about 1 to about 15, or alternatively from about 1 to about 10, microns in diameter.
  • the microsphere is a biodegradable polymer, non-limiting examples of such include poly(lactic-co-glycolic acid)("PLGA”); polycaprolactone ("PLC”); chitosan; gelatin; DNA hydrogen; acetalated dextran; poly(lactide); poly(glycolide);
  • the biodegradable polymer is poly(lactic-co-glycolic acid) or PLGA.
  • the microsphere comprises a non-biodegradable polymer.
  • non-biodegradable polymers include without limitation, of one or more of poly(ethylene vinyl acetate), poly( vinyl acetate), silicone polymers, polyurethanes, polysaccharides such as a cellulosic polymers and cellulose derivatives, acyl substituted cellulose acetates and derivatives thereof, copolymers of poly(ethylene glycol) and poly(butylene terephthalate), polystyrenes, polyvinyl chloride, polyvinyl fluoride, poly( vinyl imidazole), chorosulphonated polyolefins, polyethylene oxide, and copolymers and blends thereof.
  • the microsphere comprises a metal.
  • the metal can be selected from, but not limited to, one or more of cobalt, chromium, gold, nickel, platinum, stainless steel, titanium, tantalum, nickel-titanium, and alloys and combinations thereof.
  • compositions of the invention can be formulated as a frozen composition, e.g., flash frozen, dried or lyophilized for storage and/or transport.
  • the composition can administered alone or in combination with a carrier, such as a pharmaceutically acceptable carrier or a biocompatible scaffold.
  • a carrier such as a pharmaceutically acceptable carrier or a biocompatible scaffold.
  • Compositions of the invention may be conventionally administered rectally as a suppository, parenterally, by injection, for example, intravenously,
  • Oral formulations include such normally employed excipients such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suppositories, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain about 10% to about 95% of active ingredient, preferably about 25% to about 70%.
  • compositions are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective for the disease or condition by treated.
  • the quantity to be administered depends on the subject to be treated. Precise amounts of the composition to be administered depend on the judgment of the practitioner. Suitable regimes for initial administration and boosters are also variable, but are typified by an initial administration followed by subsequent administrations.
  • compositions In many instances, it will be desirable to have multiple administrations of the compositions about, at most about or at least about 3, 4, 5, 6, 7, 8, 9, 10 days or more.
  • the administrations will normally range from 2 day to twelve week intervals, more usually from one to two week intervals.
  • Periodic boosters at intervals of 0.5-5 years, usually two years, may be desirable to maintain the condition of the immune system
  • additional pharmaceutical compositions are administered to a subject to support or augment the compositions as described herein.
  • Different aspects of the present invention involve administering an effective amount of the composition to a subject.
  • such compositions can be administered in combination with modifiers of the immune system.
  • Such compositions will generally be dissolved or dispersed in a
  • phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, or human. As used herein,
  • “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated.
  • the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid poly(ethylene glycol), and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants.
  • the prevention of the action of undesirable microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • an effective amount of therapeutic composition is determined based on the intended goal.
  • unit dose or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and regimen.
  • the quantity to be administered depends on the result and/or protection desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above.
  • This disclosure also provides a method for preparing a composition as described herein, comprising, or alternatively consisting essentially of, or yet further consists of, the steps of admixing, contacting or culturing a biocompatible microsphere with a biofilm- generating probiotic bacterium and a prebiotic.
  • the method further comprises adding or admixing a prebiofilmic that supports the formation and growth of a biofilm by the bacterium.
  • Non-limiting examples of such include, one or more of a DNA binding polypeptide or protein and/or a DNABII polypeptide or protein.
  • the prebiotic utilized in the method comprises a water-soluble carbohydrate, which can be selected from, but not limited to, one or more of inulin, oligofructose, fructo-oligosaccharide, galacto-oligosaccharide, glucose, maltose, maltodextrins, polydextrose, sucrose, fructose, lactose, isomaltulose, polyols, glycerol, and combinations thereof.
  • a water-soluble carbohydrate which can be selected from, but not limited to, one or more of inulin, oligofructose, fructo-oligosaccharide, galacto-oligosaccharide, glucose, maltose, maltodextrins, polydextrose, sucrose, fructose, lactose, isomaltulose, polyols, glycerol, and combinations thereof.
  • a method for treating or preventing a disease in a subject comprising administering to a subject an effective amount of a composition as described above, to a subject in need of such treatment.
  • a subject intends an animal (e.g., murine, bovine, canine, feline, equine, simian) or a human.
  • Non-limiting diseases to be treated include, but not limited to, psychological disorders, such as depression or anxiety, enteric infectious disease, infection-induced colitis, traveler's diarrhea, inflammatory bowel disease (IBD), colitis, diarrheal illness, vaginosis, wound, burns, psoriasis, dermatitis, tooth decay, periodontitis, sinusitis, or any of chronic and/or recurrent disease that is caused by pathogenic bacteria displacing healthy bacteria or nectrotizing enterocolitis (NEC).
  • the compositions can be administered to support antibacterial immunity, enhancing or supporting the gastrointestinal barrier, or antagonizing disease-related bacterial infection.
  • the disease is vaginosis.
  • the disease is colitis or traveler's diarrhea.
  • the composition is specifically selected for the disease to be treated.
  • the composition further comprises a prebiofilmic.
  • the prebiofilmic comprises a DNA binding polypeptide or protein and/or a DNABII polypeptide or protein, e.g., HU, a fragment thereof and/or an equivalent of each thereof.
  • the composition is administered as a suppository.
  • the composition of the method is administered to provide from about 1 x 10 7 to about 1 x 10 9 CFU/ml of the bio film-generating probiotic bacterium. In some embodiments, the composition is administered at about 6, 12, 18, 24, 36, 48, and 72 hours. In some embodiments, the composition is administered in a single dose.
  • a method of administering a probiotic comprising administering a dose of a composition as described above, comprising, or alternatively consisting essentially of, or yet consisting of, a biocompatible microsphere, a biofilm-generating probiotic bacterium, a prebiotic, and a prebiofilmic to a subject in need of such treatment.
  • the composition of the method is administered to provide from about 1 x 10 7 to about 1 x 10 9 CFU/ml of the biofilm-generating probiotic bacterium.
  • the composition is administered at about 6, 12, 18, 24, 36, 48, and 72 hours.
  • the composition is administered in a single dose. Kits
  • kits containing one or more compositions as described herein comprises, or alternatively consists essentially of, or yet further consists of, a composition as described above, and instructions for use.
  • the kit comprises a microsphere and instructions to make the composition as described above.
  • the bacteria and prebiotic are also provided in the kit.
  • L. reuteri was introduced via oral gavage, but instead of repeating the gavage daily, which is typically needed for retention of planktonic bacteria and for beneficial effects 15, 41, a single administration of L. reuteri was provided.
  • reuteri were grown in biofilm cultures or biofilm grown on poly(lactic-co-glycolic acid) microspheres, such as PLGA, or other FDA approved and biodegradable microspheres (hydrolyzed into lactic acid and glycolic acid) with diameters ranging from 20-300 ⁇ (Beer, et al., (1998) Poly (Lactic-Glycolic) Acid Copolymer Encapsulation of Recombinant Adenovirus Reduces Immunogenicity in Vivo, Gene Ther, 5: 740-6; Kumari, et al, (2010) Biodegradable Polymeric Nanoparticles Based Drug Delivery Systems, Colloids Surf B Biointerfaces, 75:1-18).
  • poly(lactic-co-glycolic acid) microspheres such as PLGA, or other FDA approved and biodegradable microspheres (hydrolyzed into lactic acid and glycolic acid) with diameters ranging from 20-300 ⁇ (Beer, et al., (1998) Poly (Lactic-Glycolic)
  • C. rodentium treated with Z. reuteri biofilm showed a >2 fold decrease in CFU/ml compared to untreated (Table 1), regardless of the state of the introduced L. reuteri. More interesting, while all the L. reuteri proliferated during the 16 hour challenge, the L. reuteri introduced in the form of a biofilm yielded > 10-fold more CFUs than when added in planktonic form.
  • Biofilms were treated with 10 CFU of challenge condition
  • This example has provided strong evidence that the probiotics in the biofilm state provides a superior formulation to bacteria grown planktonically. It also provides one example of how to prepare these biofilms including the frequency of dosing. In addition, the example examines the nature of the biofilm itself to begin to determine why this state out performs planktonic bacteria. Finally, it examines the shelf life of the preparations as a prelude to reduction to practice in human hosts. Combined, this example identifies and characterizes the conditions and constituents for probiotic biofilm preparations.
  • L. reuteri forms a robust biofilm in vitro and that L. reuteri in a 24-hour biofilm establishes well in the mouse gut were shown. This Example varies the age of the biofilm to determine the optimal age for biofilm establishment.
  • L. reuteri In vivo L. reuteri biofilms. L. reuteri begins to attach almost immediately when exposed to a surface. After 6 hours sufficient biomass has been produced to be both visible and to start forming the classic biofilm structures (e.g., mushroom, Abee and Kuipers, (201 1) Understanding Microbial Behavior within and Outside the Host to Improve Food
  • L. reuteri biofilms are isolated at about 6, 12, 18, 24, 36, 48 and 72 hours, that have been grown on PGLA microspheres with HU and calf thymus DNA (as described above) normalizing to CFUs (108) and introducing them by gavage into mice (9 per time point from triplicate experiments). Each mouse is assessed by counting total lactobacilli levels in fecal samples daily for 12 days (cultured on MRS agar).
  • this Example uses a real-time PCR method to assess 16S rRNA gene sequence copy numbers for the Lactobacillus genus (including some species of Shoeslla, Pediococcus, and Leuconostoc due to difficulties with primer specificity) and specifically for L. reuteri.
  • the 16S rRNA gene copy numbers is determined in the feces daily for 12 days, as well as in the colon, cecum, small intestine (including ileum, jejunum, and duodenum), and stomach (including the forestomach) using real-time PCR on Days 1, 3, 6, and 12 post-oral inoculation. Sham mice with and without planktonic cells serve as controls.
  • a significant increase in L. reuteri levels in mice treated with biofilm-grown L. reuteri in comparison to sham or planktonic-treated mice is an indicator of durability and robustness.
  • L. reuteri grows in each to varying degrees.
  • the Example also varies the starting pH to about 5.5, 6, 6.5 or 7 as L. reuteri growth is favored under more acidic conditions. While L. reuteri can be grown microaerophilically under 5% C02, stressful conditions of times favor biofilm growth (Flemming, and Wingender, (2010) The Biofilm Matrix, Nat Rev Microbiol, 8:623-33); here L.
  • reuteri biofilms are also grown in air or in the absence of oxygen (anaerobic chamber).
  • the Example varies the prebiofilmics of HU (about 0.1, 1, 10, 100 ⁇ g/ml) and calf thymus DNA (about 0.1, 1, 10, 100 ⁇ g/ml). All the aforementioned biofilms are assessed by CSLM with LIVE/DEAD® staining in triplicate for height, average thickness and biomass as indicators of robust growth.
  • L. reuteri Dosing of L. reuteri; frequency and size. Rhe frequency and or size of dosing improves the durability and robustness of the introduction of L. reuteri are determined.
  • L. reuteri biofilms are grown on PLGA microspheres with added HU and calf thymus DNA for 24 hours (or an age condition as determined in Example 4.1 and 4.2).
  • L. reuteri biofilms are introduced to mice by oral gavage creating a matrix of varying the dose (10 7 , 10 8 and 10 9 CFUs) as well as the frequency (single dose, or daily dose up to 3 days) yielding 9 different conditions.
  • L. reuteri levels are assessed in vivo on Days 1, 3, 6, and 12 post gavage as outlined in Example 4.1. Nine mice (from triplicate experiments) for each condition at each time point are used. Sham mice with and without planktonic cells serve as controls.
  • L. reuteri biofilms can be dispersed by antisera to a DNABII family member (e.g., E. coli IHF).
  • a DNABII family member e.g., E. coli IHF
  • this Example tests the bacteria released (dispersed) due to anti-IHF treatment.
  • 24 hour L. reuteri biofilms (no added PLGA, HU or DNA so as to facilitate dispersal) grown in chamber slides are treated with anti-IHF20. As the peak of dispersal is about 8 to 12 hours after treatment (Goodman, et al.
  • Biofilms are found to be superior for establishment, persistence and duration of probiotic bacteria in the gut. It is not the biofilm per se that possesses superior features to planktonic bacteria but the bacteria that are dispersed from biofilms. In effect, the biofilm would be acting as a dispersed-bacteria generator. Indeed, physiologic differences in dispersed bacteria as compared to laboratory grown planktonic bacteria (e.g. in antibiotic sensitivity) have been observed.
  • L. reuteri preparations need to be in a sufficiently stable form.
  • L. reuteri biofilms have been flash frozen and found no diminution in CFUs and minimum inhibitory concentration or MIC (>2 mg/ml ampicillin; MIC for planktonic L. reuteri ⁇ 4 ⁇ g/ml) suggesting L. reuteri retains at least one property of its biofilm state, enhanced MIC.
  • Optimized L. reuteri biofilms (Example 4.1. to 4.3) for ambient air freezing to -20°C and -80°C with and without glycerol (a cryo-protectant; See also Example 2) as well as flash freezing to -80°C (placing storage tubes with fresh bacterial suspensions in dry ice-ethanol) are examined.
  • Example 4.1 Media are first removed and the resulting biofilm are scraped off and treated to freeze. Bacteria are stored at these temperatures for 1 day, 1 week or 1 month and then thawed at ambient room temperature to be used for introduction into mice by oral gavage. Nine mice from triplicate experiments are used with a similar number for controls using planktonic bacteria and optimized biofilm bacteria (Example 4.1 to 4.3). Each mouse is assessed as in Example 4.1. [0145] Desiccation. Optimized L. reuteri biofilms (Example 4.1. to 4.3) via lyophilization after freezing using the optimized technique in Example 4.5 are examined.
  • Desiccated bacteria are stored at room temperature for about 1 day, 1 week or 1 month and then rehydrated with the original biofilm volume of sterile distilled water at ambient room temperature to be used for introduction into mice by gavage.
  • Nine mice from triplicate experiments are used with a similar number for controls using planktonic bacteria and optimized biofilm bacteria (Example 4.1 to 4.3). Each mouse is assessed as in Example 4.1.
  • L. reuteri ATCC23272
  • Additional strains of L. reuteri e.g. strain 100-23, ATCCPTA6475, ATCC55730
  • L. reuteri strains that are commercially available (Fleet® Pedia-LaxTM Probiotic YumsTM ⁇ lOOmillion CFU/tablet, L. reuteri Protectis®DSM 17938 and Gerber® Soothe Colic Drops ⁇ 100 million CFU/serving (5 drops, ⁇ 200ul), L. reuteri Protectis®DSM 17938) are examined. This Example finds that by dissolving each product in water and using them directly in in vitro competition experiments with C.
  • each product is shown to be no better than the strain of L. reuteri in planktonic form.
  • microspheres as well as inherent cargo that may facilitate either probiotic growth or inhibit pathogens are explored.
  • PLGA microspheres are utilized as a surface to grow the biofilms, there are other FDA approved or GRAS biodegradable microspheres that may prove advantageous for the goals. As shown in Table 2, 5 additional types of microspheres are examined (F. Chellat, et al., (2000) In Vitro and in Vivo Biocompatibility of Chitosan-Xanthan Polyionic Complex, J Biomed Mater Res., 51 : 107-16; D. Costa, et al, (2012) Swelling Behavior of a New
  • DNA (hydrogel) e ' f Variable DNA, ethylene glycol diglycidyl ether
  • the cargo of PLGA is known to diffuse slowly or not even at all relative to the rate of microsphere hydrolysis (Fredenberg, et al. (2011) The Mechanisms of Drug Release in Poly(Lactic-Co-Glycolic Acid)-Based Drug Delivery Systems—a Review, Int J Pharm, 415:34-52).
  • microspheres with prebiotic cargo were synthesized and evaluated for their ability to support L. reuteri growth in vitro and in vivo in the mouse models.
  • microspheres These cargos include, but not limited to, inulin, fructo-oligosaccharides, and galacto-oligosaccharides as they support lactobacilli growth.
  • microspheres with MRS media and/or glycerol are made, as the former is restrictive to Gram-negative bacteria some of which are pathogens and the latter stimulates reuterin production (an antimicrobial molecule believed to give L. reuteri an advantage against competing bacteria).
  • L. reuteri biofilm growth on these microspheres is performed on the conditions observed in Example 4 (or Example 5.1 with a variant microsphere) and is adjudicated by CSLM for height, thickness and biomass.
  • This example tests prebiofilmics in vitro.
  • prebiofilmics HU and DNA
  • PLGA microspheres and the microsphere types from Example 5.1
  • biofilms are grown under the conditions observed in Example 4 with microspheres synthesized in the presence of HU and or DNA (so as to be encapsulated in the interior of the microsphere) and are adjudicated by CSLM for height, thickness and biomass.
  • This example tests a combination of prebiotics and prebiofilmics in vitro.
  • a matrix of combinations of the two probiotic and two prebio filmic cargos is created (all 16 combinations of two, all 4 combinations of 3, and the single combination of all 4 equaling 21 total combinations) to find the suitable prebiotics or prebiofilmics.
  • biofilms are grown under the conditions observed in Example 1 with PLGA microspheres (and the microsphere types from Example 5.1) synthesized in the presence of cargo and are adjudicated by CSLM for height, thickness and biomass.
  • This example tests optimized components in vivo. Conditions from Example 5.2 that yielded the biofilms are used for in vivo experiments. The four most promising conditions for PLGA microsphere cargo (or the two most promising PLGA and two most promising other type of microsphere from Example 5.1) are tested on nine mice each derived from triplicate experiments. Each mouse is assessed as in Example 4.1 on Days 1, 3, 6, and 12 post-Z. reuteri introduction. Sham mice (no bacteria) and planktonic bacteria serve as controls.
  • Microspheres containing various probiotic cargos to determine if they support pathogen biofilm growth are examined.
  • the microspheres containing prebiofilmics come into contact with a pathogen (i.e., C. rodentium strain DBS120 (pCRPl ::Tn5)) as well as probiotic.
  • a pathogen i.e., C. rodentium strain DBS120 (pCRPl ::Tn5)
  • This example tests pathogen impeding nutrients in vitro.
  • C. rodentium is grown in LB media and used to seed biofilms with PLGA and the
  • Biofilms is adjudicated by CSLM for height, thickness and biomass compared to empty PLGA microspheres.
  • This example tests pathogen impeding nutrients in vivo. Taking into consideration the results from in vitro biofilm data in Example 5.3, four cargos for C. rodentium biofilm growth and use them in vivo in mouse models are examined. Nine mice for each condition per time point (from triplicate experiments) are used with planktonic C. rodentium and sham (no bacteria) as controls. C. rodentium levels in the stool is determined via culture on all days post oral C. rodentium administration. On Days 1, 6, 12 and 24 post-oral C.
  • the colon is removed and transected longitudinally so that inflammatory cytokines ⁇ e.g., TNF-a), inflammatory mediators ⁇ e.g., inducible nitric oxide synthase (iNOS)), and chemokines (e.g., CCL2) can be assessed in half of the colon via real-time RT- PCR.
  • inflammatory cytokines ⁇ e.g., TNF-a
  • inflammatory mediators ⁇ e.g., inducible nitric oxide synthase (iNOS)
  • chemokines e.g., CCL2
  • immunohistochemistry is used to assess leukocyte infiltration into the colon ⁇ e.g., F4/80+ macrophages; myeloperoxidase (MPO)+
  • microsphere bio film preparations can include alternative types of microspheres and varying cargo. It is Applicants' belief that bio films (regardless of surface) are superior to planktonic bacteria at seeding probiotic colonization in vivo.
  • Non-limiting examples cargos include without limitation specific effectors of innate immunity that reduce inflammation, part of the process leading to dysbiosis.
  • microspheres can comprise conditioned media from L. reuteri as L. reuteri produce such substances.
  • other bacteria are within the scope of this disclosure, e.g., C. rodentium and L. reuteri, in general for pathologies due to dysbiosis.
  • Controls include each bacterial species without the other under each condition (e.g. , the addition of C. rodentium added to PLGA microspheres without L. reuteri in each chamber slide). All experiments are done in triplicate. Table 3. Systematic Approach To Find Optimal Conditions For L. Reuteri Vs. C. Rodentium Challenge
  • L. reuteri biofilm preparations for introduction into animals are prioritized based on the greatest retention or supremacy of L. reuteri observed.
  • L. reuteri is prepared based on any successes derived from Examples 4.1, 4.4 and 4.5.
  • L. reuteri biofilms are introduced 12 hours prior to oral challenge with C. rodentium.
  • Triplicate experiments are conducted for a final sample size of 9 mice for each condition and time point that are assessed at 1, 6, 12, and 24 days post-challenge (peak C. rodentium infection occurs at about Day 12).
  • C. rodentium levels in the stool are assessed and pathogen-induced colitis is assessed as in Example 5.3.
  • L. reuteri levels are also assessed as in Example 4.1.
  • controls include C. rodentium without L. reuteri and C. rodentium challenge plus planktonic L. reuteri.
  • rodentium (about 12, 24 or 36 hours after the final L. reuteri treatment).
  • mice from triplicate experiments.
  • Vehicle mice infected with C. rodentium and single planktonic L. reuteri serve as controls.
  • C. rodentium levels and pathogen-induced colitis are assessed on Days 1, 6, 12 and 24 post-challenge as in Example 5.3, with L. reuteri levels assessed as in Example 4.1.
  • Controls include each bacterial species without the other under each condition ⁇ e.g. , the addition of L. reuteri to chamber slides without extant C. rodentium). All experiments are done in triplicate.
  • Challenge of C. rodentium by L. reuteri bio films in vivo Here this Example determines if L. reuteri biofilms can challenge prior C. rodentium infection in the murine model. Three different C. rodentium conditions (single gavage 12, 24 or 36 hours) prior to challenge with L. reuteri were examined. Four L. reuteri biofilm conditions including dosing (Example 6.2) are used to challenge C. rodentium. At least two of these conditions are derived from Example 6.1. Nine mice from triplicate experiments are used to test each of these 12 conditions. Pathogen-induced colitis is assessed as in Example 5.3, with L. reuteri levels assessed as in Example 4.1.
  • this Example determines how effective L. reuteri introduced in the form of a biofilm is as a prophylactic to C. rodentium challenge and as a treatment for extant C.
  • Pathogens included in an in vitro survey are enteric pathogens with different modes of infection, including invasive pathogens ⁇ e.g. , Salmonella enterica subspecies Typhimurium and Shigella flexneri), additional A/E pathogens (e.g. ,
  • Enterohemorrhagic E. coli 0157:H7; and Enteropathogenic E. coli), and toxin-producing pathogens e.g., Vibrio cholera and Enterotoxigenic E. coli
  • the rate limiting step in these experiments is finding co-culturing conditions that sufficiently mimic the in vivo state.
  • ANOVA factor analysis of variance
  • probiotic i.e., probiotic vs. vehicle control
  • condition i.e., biofilm vs. planktonic
  • time of culture i.e., 6, 12, 18, 24, or 36 hours
  • Probiotics have been widely used for digestive health benefits, although few actually prevent pathogen colonization and reduce the inflammatory response.
  • the effects of probiotic bacteria can be significantly improved by the manner in which they are introduced into the host; specifically by growing them in the form of a biofilm.
  • the data suggest that colonization in vivo by the probiotic L. reuteri is greatly enhanced when grown as a biofilm compared to planktonic-grown cells.
  • L. reuteri was grown in the presence of a biodegradable surface (PLGA), colonization was also increased indicating that the conditions were optimized that allowed a vast improvement in regards to L. reuteri establishment within the host.
  • PLGA biodegradable surface
  • Probiotic microbes have also been shown to reduce anxiety and depression in otherwise healthy humans and laboratory animals.
  • a combination of Lactobacillus helviticus and B. longum administered daily for 30 days was shown to reduce anxiety and depression in healthy human volunteers and in healthy rats (Messoudi et al. (2011) Beneficial
  • prophylactic treatment with L. reuteri biofilms is assessed to determine if C. rodentium will prevent bacterium-induced sickness, anxiety-, and depressive-like behaviors.
  • Preparations of L. reuteri biofilms that are found to be superior in in vitro assays are administered to mice via oral gavage 12 hours prior to oral challenge with C. rodentium. Triplicate experiments are conducted for a final sample size of 9 mice for each condition and time point that are assessed at 1, 6, 12, and 24 days post-challenge (peak C. rodentium infection occurs about Day 12).
  • animal behavior is assessed for locomotor activity (such as on the open field test), anxietylike behavior (such as in the ligh dark preference test and elevated plus maze), depressive- like behavior (such as on the tail suspension test and Porsolt forced swim task), and sickness behavior (such as with the sucrose preference test).
  • Blood serum cytokines associated with emotional and illness behavior e.g., IL- ⁇ / ⁇ and IL-6 are assessed on each day. Circulating corticosterone levels will also be assessed.
  • Neuronal activation in the brain, especially the paraventricular nucleus of the hypothalamus, are assessed using c-Fos immunoreactivity.
  • L. reuteri can be used as a therapeutic to treat C. rodentium -induced sickness, anxiety-like, and depressive-like behavior also is assessed. For example, the compositions are tested to determine whether treating an established C. rodentium infection will reduce sickness, anxiety-, and depressive-like behaviors. Preparations of L. reuteri that are found to be superior in in vitro assays are administered to mice via an oral gavage 12, 24, and/or 36 hours after oral challenge with C. rodentium. On days 1, 6, 12, and 24 post-C.
  • animal behavior is assessed for locomotor activity (such as on the open field test), anxiety-like behavior (such as in the ligh dark preference test and elevated plus maze), depressive-like behavior (such as on the tails suspension test and Porsolt forced swim task), and sickness behavior (such as with the sucrose preference test).
  • locomotor activity such as on the open field test
  • anxiety-like behavior such as in the ligh dark preference test and elevated plus maze
  • depressive-like behavior such as on the tails suspension test and Porsolt forced swim task
  • sickness behavior such as with the sucrose preference test.
  • Circulating cytokines associated with emotional and illness behavior e.g., IL- ⁇ / ⁇ and IL-6 are assessed on each day.
  • Circulating corticosterone levels are also assessed.
  • Neuronal activation in the brain, especially the paraventricular nucleus of the hypothalamus are assessed using c-Fos immunoreactivity.
  • Probiotic administration may be beneficial in the prevention of NEC.
  • probiotics must be administered daily to achieve beneficial effects.
  • Applicants describe herein a novel probiotic delivery system in which the probiotics are grown as a biofilm on the surface of prebiotic-loaded biocompatible microspheres, allowing enhanced and more durable efficacy with only a single treatment.
  • NEC hypoxia/hypothermia/hypertonic feeds
  • Pups were sacrificed when clinical signs of NEC developed or by 96 hours after birth.
  • a verified histologic NEC injury grading system was used to measure the incidence and severity of NEC, with Grade 2 or greater injury considered to be consistent with NEC.
  • a dessication tolerance assay was used to test stability and viability of the bacteria combined with the microspheres.
  • the assay can generally be conducted by performing the following steps. To grow the bacteria culture, transfer 1ml to al .5ml of the culture to a microcentrifuge tube (1 tube per condition per time period to be tested). Add aboutlO ⁇ of hydrated microspheres, trehalose, or nothing to the tube. Incubate the tube for 30 minutes and then pellet the cells via centrifugation. Remove the supernatant and wash the pellet twice with sterile saline. Afterwards, remove all liquid from the pellet.
  • P. fluorescens and a proprietary Azospirillum sp. were placed after 90 days incubation at 40°C while on top of Drierite, a strong desiccant, and then rehydrated and tested for viability.
  • P. fluorescens with no microspheres shows a complete loss of colony forming units (CFU) after just one week in these conditions, whereas when incubated with cellulose microspheres, there are 10 5 viable cells after 90 days in these conditions.
  • Azospirillum sp. shows significant loss of CFUs after 30 days and complete loss after 90 days when grown without the microsphere formulation; however, when stored in harsh conditions with the microspheres, 10 6 CFU/ml of Azospirillum sp. are viable even after 90 days.
  • Microspheres filled with L. reuteri growth medium as cargo were utilized to provide a surface that leaches buffered nutrients to the bacteria for the formation of a biofilm that enhances survivability at low pH.
  • Bacterial cells with microspheres show over a 2 log increase in viable colony forming units compared to cells without microspheres after sitting in pH 2 gastric acid for 4 hours.
  • L. reuteri with microspheres show increased adherence to mouse colonic cells, addressing the problem of poor colonization and sustainability of orally administered bacteria.
  • the novel microsphere formulations not only increase survivability at low pH, but also contribute to colonization of beneficial bacteria in the gut, making L. reuteri a more efficient probiotic.
  • Microspheres filled with L. reuteri growth medium as cargo were utilized to provide a surface that leaches buffered nutrients to the bacteria for the formation of a biofilm that enhances survivability at low pH.
  • Bacterial cells with microspheres show over a 2 log increase in viable colony forming units compared to cells without microspheres after sitting in pH 2 gastric acid for 4 hours.
  • L. reuteri with microspheres show increased adherence to mouse colonic cells, addressing the problem of poor colonization and sustainability of orally administered bacteria.
  • a cellular adherence assay such as that used to generate the above information, can generally be conducted by performing the following steps. First, grow up a mammalian cell culture line and dilute to ⁇ 10 6 cells/ml. Transfer 500ul of the diluted mammalian cell lines to a 48-well plate. Then, grow to confluence (time varies, at least 16 hours) and grow the bacterial culture overnight. Afterwards, transfer 500ul of the bacterial culture to a 1.5ml microcentrifuge tube (1 tube per condition per time period). Pellet the bacterial cells via centrifugation and wash the pellet 2-3 times to remove all growth medium. Resuspend the pelleted bacteria in a cell line culture medium.
  • serially dilute and plate to calculate the number of bacteria that remained adhered to the mammalian cells.
  • H. pylori 26695 Hp Hp0835 (Hup) B. burgdorferi B31 Bb BB_0232 (Hbb) Moraxella catarrhalis RH4 Mc YP_003626307 (HimA)
  • Treponema palladium Nichols Tp TP_0251 DNA binding protein II

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107267410A (zh) * 2017-05-09 2017-10-20 上海久州环保技术有限公司 一种抗菌方法
WO2017203533A1 (en) * 2016-05-25 2017-11-30 Mybiotics Pharma Ltd. Composition and methods for microbiota therapy
US20180000878A1 (en) * 2014-03-06 2018-01-04 Research Institute At Nationwide Children's Hospital Prebiotic formulations
JP2018517430A (ja) * 2015-05-11 2018-07-05 マイバイオティクス ファーマ リミテッド 腸における細菌のコロニー形成のために固体粒子上で生菌のバイオフィルムを成長させるためのシステムおよび方法
WO2018183355A1 (en) * 2017-03-27 2018-10-04 Research Institute At Nationwide Children's Hospital Prebiotic formulations
PL423964A1 (pl) * 2017-12-19 2019-07-01 Izabela Anna Zawisza Kompozycja bakterii probiotycznych, zastosowanie kompozycji bakterii probiotycznych oraz sposób personalizowana kompozycji bakterii probiotycznych
US10369176B2 (en) * 2014-03-06 2019-08-06 Research Institute At Nationwide Children's Hospital Probiotic formulations and methods for use
EP3462917A4 (de) * 2016-05-29 2020-01-08 The State of Isreal, Ministry of Agriculture & Rural Development, Agricultural Research Organization (ARO) Verfahren zur erzeugung von bakteriellen zusammensetzungen
JP2020508700A (ja) * 2017-03-03 2020-03-26 ビオミレニア ソシエテ パ アクシオンス シンプリフィエ 細胞を培養し、試験するための微粒子
US10709744B1 (en) 2019-03-28 2020-07-14 Mybiotics Pharma Ltd. Probiotic biofilm suppositories
WO2020247536A1 (en) * 2019-06-03 2020-12-10 Research Institute At Nationwide Children's Hospital Prebiotic formulations for prevention of sepsis and necroenterocolitis induced neurodevelopmental deficiencies
WO2021130647A1 (en) * 2019-12-24 2021-07-01 S A Pharmachem Private Limited Probiotic formulations for liver and oral health and enhancement of immunity
US20210290701A1 (en) * 2018-08-03 2021-09-23 Research Institute At Nationwide Children's Hospital Prenatal probiotic formulations
CN113766923A (zh) * 2019-03-28 2021-12-07 迈彼欧提克斯制药有限公司 益生菌生物膜组合物及其制备方法
US20220154134A1 (en) * 2019-03-28 2022-05-19 Mybiotics Pharma Ltd. Probiotic biofilm compositions and methods of preparing same
US11484479B2 (en) 2015-07-14 2022-11-01 Research Institute At Nationwide Children's Hospital Formulation for the elimination of cariogenic and opportunistic pathogens within the oral cavity

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3365027B1 (de) 2015-10-14 2022-03-30 Research Institute at Nationwide Children's Hospital Hu-spezifische antikörper und deren verwendeng zur hemmung von biofilm
AU2020350573A1 (en) * 2019-09-16 2022-04-07 Vedanta Biosciences, Inc. Methods and compositions for preserving bacteria
CN114196665B (zh) * 2020-09-17 2023-08-15 南京农业大学 一种用于益生菌生物膜冷藏的载体及其制备方法
CN112760247B (zh) * 2020-11-10 2022-10-25 深圳华大生命科学研究院 用于预防和/或治疗生殖道菌群紊乱和/或骨质流失引起的疾病的鼠李糖乳杆菌
KR102463565B1 (ko) * 2021-05-10 2022-11-04 주식회사 클린힐 포스트바이오틱스 혼합물이 함유된 친환경 세탁세제 조성물 및 그의 제조를 위한 세탁세제 조성물 제조 장치

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US6638369B1 (en) 2002-05-07 2003-10-28 The United States Of America As Represented By The Secretary Of The Navy Non-chromate conversion coatings
EP2450062A1 (de) 2009-07-01 2012-05-09 Universidad De Concepcion Hydrokolloidgipse mit lebensfähigen probiotischen stämmen von lactobacillus spp. für verschiedene arten von wunden, infizierten geweben, trockenen wunden und tiefen wunden
US8815276B2 (en) 2009-06-26 2014-08-26 Aarhus Universitet Three-dimensional nanostructured hybrid scaffold and manufacture thereof

Family Cites Families (68)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5549908A (en) 1993-05-20 1996-08-27 The University Of Akron Hydrolytically labile microspheres of polysaccharide crosslinked with cyanogen halide and their application in wound dressings
WO1998050018A1 (en) 1997-05-06 1998-11-12 Xiao Yu Wu Drug delivery system
US6551795B1 (en) * 1998-02-18 2003-04-22 Genome Therapeutics Corporation Nucleic acid and amino acid sequences relating to pseudomonas aeruginosa for diagnostics and therapeutics
DE19952983A1 (de) * 1999-11-03 2001-05-17 Acgt Progenomics Ag Verfahren zum Transfer von molekularen Substanzen mit prokaryontischen nukleinsäurebindenden Proteinen
US6486314B1 (en) 2000-05-25 2002-11-26 Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno Glucan incorporating 4-, 6-, and 4, 6- linked anhydroglucose units
US6887493B2 (en) * 2000-10-25 2005-05-03 Adi Shefer Multi component controlled release system for oral care, food products, nutraceutical, and beverages
WO2002077183A2 (en) * 2001-03-21 2002-10-03 Elitra Pharmaceuticals, Inc. Identification of essential genes in microorganisms
ES2345877T3 (es) * 2001-07-20 2010-10-05 Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno Usos de un glucano derivado de bacterias acido lacticas como agente contra la corrosion.
CN100478026C (zh) * 2001-09-28 2009-04-15 纽特休迪克斯公司 用于生物学组分的传递系统
US7351877B2 (en) * 2002-03-29 2008-04-01 Syngenta Participations Ag Lambda integrase mediated recombination in plants
US20050266069A1 (en) 2002-09-06 2005-12-01 Simmons Donald L Stable probiotic microsphere compositions and their methods of preparation
US9687449B2 (en) 2002-11-07 2017-06-27 Advanced Bionutrition Corp. Nutraceuticals and method of feeding aquatic animals
US7241867B2 (en) * 2003-03-06 2007-07-10 Children's Hospital, Inc. Polypeptide encoded by a nucleotide sequence of a nontypeable strain of Haemophilus influenzae genome
US8728525B2 (en) 2004-05-12 2014-05-20 Baxter International Inc. Protein microspheres retaining pharmacokinetic and pharmacodynamic properties
SG160329A1 (en) * 2005-02-18 2010-04-29 Novartis Vaccines & Diagnostic Proteins and nucleic acids from meningitis/sepsis-associated escherichia coli
JP2008546390A (ja) * 2005-06-16 2008-12-25 ネイションワイド チルドレンズ ホスピタル， インコーポレイテッド 分類不能型インフルエンザ菌の中耳炎単離物の遺伝子
US8906392B2 (en) * 2005-12-16 2014-12-09 University Of Kansas Nanocluster compositions and methods
CA2644968A1 (en) * 2006-03-07 2007-09-13 Nestec S.A. Synbiotic mixture
US20100316769A1 (en) 2006-03-29 2010-12-16 Gail Czarnecki-Maulden Dietary supplements containing probiotics
US20070286822A1 (en) 2006-06-12 2007-12-13 Anacor Pharmaceuticals Inc. Compounds for the Treatment of Periodontal Disease
US20100303723A1 (en) 2006-11-20 2010-12-02 Massachusetts Institute Of Technology Drug delivery systems using fc fragments
BRPI0809454A2 (pt) * 2007-03-27 2014-09-09 Procter & Gamble Métodos e kits para a administração de probióticos
EP2022502A1 (de) * 2007-08-10 2009-02-11 Nestec S.A. Lactobacillus rhamnosus und Gewichtssteuerung
IL199781A0 (en) * 2009-07-09 2010-05-17 Yohai Zorea Heat resistant probiotic compositions and healthy food comprising them
UA106925C2 (uk) * 2008-11-14 2014-10-27 Юністро Холдінгс Пте Лтд Пробіотичні композиції, способи та пристрій для їх введення
IT1393931B1 (it) * 2009-02-25 2012-05-17 Neuroscienze Pharmaness S C A R L Composizione di spore di batteri non patogeni
ES2643148T3 (es) * 2009-05-26 2017-11-21 Advanced Bionutrition Corporation Composición en polvo seco estable que comprende microorganismos biológicamente activos y/o materiales bioactivos y métodos de producción
CA2769636A1 (en) 2009-07-30 2011-02-03 The Procter & Gamble Company Oral care articles and methods
IE20090662A1 (en) 2009-08-31 2011-08-31 Patrick T Prendergast Cariogenesis, halitosis, gingivitis and periodontitis treatment and preventive compositions.
US20110070334A1 (en) * 2009-09-20 2011-03-24 Nagendra Rangavajla Probiotic Stabilization
JP5415232B2 (ja) * 2009-11-10 2014-02-12 ビオフェルミン製薬株式会社 生菌製剤の製造方法
MX2012005584A (es) * 2009-11-13 2012-09-12 Unistraw Patent Holdings Ltd Composiciones probioticas, metodos y aparatos para su administracion.
JP5592682B2 (ja) * 2010-03-26 2014-09-17 株式会社明治 少容量に充填したプロバイオティクス含有組成物
US8999291B2 (en) * 2010-03-29 2015-04-07 University Of Southern California Compositions and methods for the removal of biofilms
US20120128701A1 (en) 2010-09-09 2012-05-24 Nationwide Children's Hospital, Inc. Compositions and methods for the removal of biofilms
BE1019142A3 (fr) 2011-01-21 2012-03-06 Vesale Pharma S A Substance probiotique microencapsulee.
WO2012106260A2 (en) 2011-02-01 2012-08-09 Channel Medsystems, Inc. Methods and apparatus for cyrogenic treatment of a body cavity or lumen
EP2680866A1 (de) * 2011-03-01 2014-01-08 Quorum Innovations, LLC Materialien und verfahren zur behandlung von leiden in zusammenhang mit pathogenen biofilm
MX2014000458A (es) 2011-07-12 2014-05-01 Nestec Sa Probioticos para la administracion a mamiferos jovenes saludables durante el periodo de destete para mejorar l atolerancia a productos alimenticios de introduccion reciente.
FR2984352B1 (fr) * 2011-12-16 2018-02-16 Lesaffre Et Compagnie Composition comprenant une biomasse microbienne active
RU2014129910A (ru) 2011-12-21 2016-02-10 Колгейт-Палмолив Компани Композиции для ухода за полостью рта
EP2609814A1 (de) 2011-12-30 2013-07-03 Nestec S.A. Lactobacillus reuteri DSM 17938 zur Entwicklung der kognitiven Funktion
EP3659598A1 (de) 2012-06-04 2020-06-03 Gaurav Agrawal Zusammensetzungen und verfahren zur behandlung von morbus crohn und verwandten erkrankungen und infektionen
WO2014070769A1 (en) 2012-10-29 2014-05-08 The University Of North Carolina At Chapel Hill Methods and compositions for treating mucosal tissue disorders
EP2914275A1 (de) 2012-10-30 2015-09-09 Nestec S.A. Zusammensetzungen mit mikropartikeln und probiotika zur freisetzung einer synergistischen immunwirkung
JP6133068B2 (ja) 2013-01-30 2017-05-24 カンタツ株式会社 撮像レンズ
US10973861B2 (en) 2013-02-04 2021-04-13 Seres Therapeutics, Inc. Compositions and methods
US11185562B2 (en) * 2013-02-04 2021-11-30 Seres Therapeutics, Inc. Compositions and methods for inhibition of pathogenic bacterial growth
CN105307654B (zh) 2013-04-30 2022-06-07 托马斯·朱利叶斯·波洛迪 用于治疗微生物区系相关的精神病况和疾病的组合物和方法
US20160120915A1 (en) 2013-06-10 2016-05-05 New York University Methods for manipulating immune responses by altering microbiota
WO2014201305A1 (en) * 2013-06-13 2014-12-18 University Of Southern California Compositions and methods for the treatment of burkholderia infections
US20160194384A1 (en) * 2014-06-12 2016-07-07 Research Institute At Nationwide Children's Hospital Compositions and methods for the removal of biofilms
DE202013103204U1 (de) * 2013-07-17 2013-07-26 Pharmaceutical Biotechnology S.R.O. Präparat, welches eine probiotische Kultur enthält
US10203329B2 (en) 2013-09-12 2019-02-12 The Johns Hopkins University Biofilm formation to define risk for colon cancer
US9745366B2 (en) 2013-09-23 2017-08-29 University Of Southern California Compositions and methods for the prevention of microbial infections
US10233234B2 (en) * 2014-01-13 2019-03-19 Trellis Bioscience, Llc Binding moieties for biofilm remediation
US20150197558A1 (en) * 2013-09-26 2015-07-16 Trellis Bioscience, Llc Binding moieties for biofilm remediation
US20150086561A1 (en) * 2013-09-26 2015-03-26 Trellis Bioscience, Llc Binding moieties for biofilm remediation
US9596861B2 (en) 2013-12-24 2017-03-21 Sami Labs Limited Method of producing partially purified extracellular metabolite products from Bacillus coagulans and biological applications thereof
AU2015227075A1 (en) * 2014-03-06 2016-09-22 Ohio State Innovation Foundation Probiotic formulations and methods for use
US10624934B2 (en) * 2014-03-06 2020-04-21 Research Institute At Nationwide Children's Hospital Prebiotic formulations
US9652745B2 (en) 2014-06-20 2017-05-16 Hirevue, Inc. Model-driven evaluator bias detection
PL3209307T3 (pl) * 2014-10-21 2023-05-22 Aquilon Cyl Sociedad Limitada Kompozycje probiotyczne i prebiotyczne
BR112017005612A2 (pt) 2014-10-29 2017-12-12 Nestec Sa uso de l. reuteri para recuperação da disbiose da microbiota na fase inicial da vida
CN113730442A (zh) 2014-10-31 2021-12-03 潘德勒姆治疗公司 与病症的微生物治疗和诊断有关的方法和组合物
MA41020A (fr) * 2014-11-25 2017-10-03 Evelo Biosciences Inc Compositions probiotiques et prébiotiques, et leurs procédés d'utilisation pour la modulation du microbiome
EP3328215B1 (de) 2015-07-29 2021-07-07 Advanced BioNutrition Corp. Stabile trockene probiotische zusammensetzungen für spezielle diätverwendungen
WO2020028871A1 (en) 2018-08-03 2020-02-06 Research Institute At Nationwide Children's Hospital Prenatal probiotic formulations

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4683195B1 (de) 1986-01-30 1990-11-27 Cetus Corp
US6638369B1 (en) 2002-05-07 2003-10-28 The United States Of America As Represented By The Secretary Of The Navy Non-chromate conversion coatings
US8815276B2 (en) 2009-06-26 2014-08-26 Aarhus Universitet Three-dimensional nanostructured hybrid scaffold and manufacture thereof
EP2450062A1 (de) 2009-07-01 2012-05-09 Universidad De Concepcion Hydrokolloidgipse mit lebensfähigen probiotischen stämmen von lactobacillus spp. für verschiedene arten von wunden, infizierten geweben, trockenen wunden und tiefen wunden

Non-Patent Citations (103)

* Cited by examiner, † Cited by third party
Title
"Antibodies, A Laboratory Manual", 1999
"Bergey's Manual of Determinative Bacteriology", 1994, WILLIAMSWILKINS
"Current Protocols in Molecular Biology", 2007
"Current Protocols in Molecular Biology", vol. 30, 1987
"Gene Transfer and Expression in Mammalian Cells", 2003
"Gene Transfer Vectors for Mammalian Cells", 1987, COLD SPRING HARBOR LABORATORY
"Health and Nutritional Properties of Probiotics in Food Including Powdered Milk with Live Bacteria", 2001, FOOD AND AGRICULTURE ORGANIZATION
"Health and Nutritional Properties ofProbiotics in Food Including Powdered Milk with Live Bacteria", 2001, FOOD AND AGRICULTURE ORGANIZATION
"Immunochemical Methods in Cell and Molecular Biology", 1987, ACADEMIC PRESS
"Methods in Enzymology", ACADEMIC PRESS, INC.
"Molecular Cloning: A Laboratory Manual", 2001
"Nucleic Acid Hybridization", 1984
"Oligonucleotide Synthesis", 1984
"Remington's Pharmaceutical Sciences", MACK PUBLISHING COMPANY
"Transcription and Translation; Immobilized Cells and Enzymes", 1986, IRL PRESS
"Weir's Handbook of Experimental Immunology", 1996
A. KUMARI ET AL.: "Biodegradable Polymeric Nanoparticles Based Drug Delivery Systems", COLLOIDS SURF B BIOINTERFACES, vol. 75, 2010, pages 1 - 18
A. R. MACKOS ET AL.: "Probiotic Lactobacillus Reuteri Attenuates the Stressor-Enhanced Severity ofCitrobacter Rodentium Infection", INFECT IMMUN, vol. 81, 2013, pages 3253 - 63
ABEE; KUIPERS: "Understanding Microbial Behavior within and Outside the Host to Improve Food Functionality and Safety", CURR OPIN BIOTECHNOL, vol. 22, 2011, pages 133 - 5
ANDERSON, NUCLEIC ACID HYBRIDIZATION, 1999
BEER ET AL.: "Poly (Lactic-Glycolic) Acid Copolymer Encapsulation of Recombinant Adenovirus Reduces Immunogenicity in Vivo", GENE THER, vol. 5, 1998, pages 740 - 6
BORENSHTEIN, M. ET AL.: "Utility of the Citrobacter rodentium Infection Model in Laboratory Mice", CURR OPIN GASTROENTEROL, vol. 24, 2008, pages 32 - 7
C. CHASSARD, M. ET AL.: "Functional Dysbiosis within the Gut Microbiota of Patients with Constipated-Irritable Bowel Syndrome", ALIMENT PHARMACOL THER, vol. 35, 2012, pages 828 - 38
C. LUPP; M. L. ROBERTSON ET AL.: "Host-Mediated Inflammation Disrupts the Intestinal Microbiota and Promotes the Overgrowth of Enterobacteriaceae", CELL HOST MICROBE, vol. 2, 2007, pages 119 - 29
C. P. BRAEGGER; T. T. MACDONALD: "Immune Mechanisms in Chronic Inflammatory Bowel Disease", ANN ALLERGY, vol. 72, 1994, pages 135 - 41
CARROLL ET AL.: "Alterations in Composition and Diversity of the Intestinal Microbiota in Patients with Diarrhea-Predominant Irritable Bowel Syndrome", NEUROGASTROENTEROL MOTIL., vol. 24, 2012, pages 521 - 30
CHASSARD, M. ET AL.: "Functional Dysbiosis within the Gut Microbiota of Patients with Constipated-Irritable Bowel Syndrome", ALIMENT PHARMACOL THER., vol. 35, 2012, pages 828 - 38
D. BORENSHTEIN ET AL.: "Utility of the Citrobacter Rodentium Infection Model in Laboratory Mice", CURR OPIN GASTROENTEROL, vol. 24, 2008, pages 32 - 7
D. COSTA ET AL.: "Swelling Behavior of a New Biocompatible Plasmid DNA Hydrogel", COLLOIDS SURF B BIOINTERFACES, vol. 92, 2012, pages 106 - 12
D. COSTA, J. ET AL.: "Swelling Behavior of a New Biocompatible Plasmid DNA Hydrogel", COLLOIDS SURF B BIOINTERFACES, vol. 92, 2012, pages 106 - 12
EATON, A. ET AL.: "Probiotic Lactobacillus reuteri Ameliorates Disease Due to Enterohemorrhagic Escherichia coli in Germfree Mice", INFECT IMMUN., vol. 79, 2011, pages 185 - 91
F. CHELLAT ET AL.: "In Vitro and in Vivo Biocompatibility of Chitosan-Xanthan Polyionic Complex", J BIOMED MATER RES., vol. 51, 2000, pages 107 - 16
F. CHELLAT, M. ET AL.: "In Vitro and in Vivo Biocompatibility of Chitosan-Xanthan Polyionic Complex", J BIOMED MATER RES, vol. 51, 2000, pages 107 - 16
F. ITO, H. ET AL.: "Factors Affecting the Loading Efficiency of Water-Soluble Drugs in Plga Microspheres", COLLOIDS SURF B BIOINTERFACES, vol. 61, 2008, pages 25 - 9
F. TOPUZ; 0. OKAY: "Formation of Hydrogels by Simultaneous Denaturation and CrossLinking of DNA", BIOMACROMOLECULES, vol. 10, 2009, pages 2652 - 61
FLEMMING; WINGENDER: "The Biofilm Matrix", NAT REV MICROBIOL, vol. 8, 2010, pages 623 - 33
FREDENBERG ET AL.: "The Mechanisms of Drug Release in Poly(Lactic-Co-Glycolic Acid)-Based Drug Delivery Systems--a Review", INT J PHARM, vol. 415, 2011, pages 34 - 52
FRESHNEY: "Culture of Animal Cells: A Manual of Basic Technique", 2005
G. A. PREIDIS ET AL.: "Probiotics Stimulate Enterocyte Migration and Microbial Diversity in the Neonatal Mouse Intestine", FASEB J, vol. 26, 2012, pages 1960 - 9
GOODMAN ET AL.: "Biofilms Can Be Dispersed by Focusing the Immune System on a Common Family of Bacterial Nucleoid-Associated Proteins", MUCOSAL IMMUNOL, vol. 4, 2011, pages 625 - 37
GULOKSUZ ET AL.: "Depressive symptoms in Crohn's disease: relationship with immune activation and tryptophan availability", PLOS ONE, vol. 8, no. 3, 2013, pages E6043
H. C. FLEMMING; J. WINGENDER: "The Biofilm Matrix", NAT REV MICROBIOL, vol. 8, 2010, pages 623 - 33
HEMARAJATA; VERSALOVIC: "Effects of Probiotics on Gut Microbiota: Mechanisms of Intestinal Immunomodulation and Neuromodulation", THERAP ADV GASTROENTEROL, vol. 6, 2013, pages 39 - 51
J. C. DE MAN, M. ET AL.: "A Medium for the Cultivation of Lactobacilli", J APPLIED BACTERIOLOGY, vol. 23, 1960, pages 130 - 35
J. E. GUSTAVE ET AL.: "Targeting Bacterial Integration Host Factor to Disrupt Biofilms Associated with Cystic Fibrosis", J CYST FIBROS, vol. 12, 2013, pages 384 - 9
J. P. NATARO; J. B. KAPER: "Diarrheagenic Escherichia Coli", CLIN MICROBIOL REV, vol. 11, 1998, pages 142 - 201
JONES; VERSALOVIC: "Probiotic Lactobacillus reuteri Biofilms Produce Antimicrobial and Anti-Inflammatory Factors", BMC MICROBIOL, vol. 9, 2009, pages 35
K. A. BRANDSTETTER ET AL.: "Antibodies Directed against Integration Host Factor Mediate Biofilm Clearance from Nasopore", LARYNGOSCOPE, 2013
K. A. EATON, A. ET AL.: "Probiotic Lactobacillus Reuteri Ameliorates Disease Due to Enterohemorrhagic Escherichia Coli in Germfree Mice", INFECT IMMUN, vol. 79, 2011, pages 185 - 91
K. J. KAUFFMAN, C. ET AL.: "Synthesis and Characterization of Acetalated Dextran Polymer and Microparticles with Ethanol as a Degradation Product", ACS APPL MATER INTERFACES, vol. 4, 2012, pages 4149 - 55
K. MACHIELS ET AL.: "A Decrease of the Butyrate-Producing Species Roseburia Hominis and Faecalibacterium Prausnitzii Defines Dysbiosis in Patients with Ulcerative Colitis", GUT, 2013
KAUFFMAN ET AL.: "Synthesis and Characterization of Acetalated Dextran Polymer and Microparticles with Ethanol as a Degradation Product", ACS APPL MATER INTERFACES, vol. 4, 2012, pages 4149 - 55
KOO; WANKAT, KOREAN BIOCHEM. J., vol. 21, no. 1, 1988
KUMARI ET AL.: "Biodegradable Polymeric Nanoparticles Based Drug Delivery Systems", COLLOIDS SURF B BIOINTERFACES, vol. 75, 2010, pages 1 - 18
L. A. NOVOTNY ET AL.: "Structural Stability of Burkholderia Cenocepacia Biofilms Is Reliant on Edna Structure and Presence of a Bacterial Nucleic Acid Binding Protein", PLOS ONE, vol. 8, 2013, pages E67629
L. A. SARMIENTO-RUBIANO ET AL.: "Dietary Supplementation with Sorbitol Results in Selective Enrichment of Lactobacilli in Rat Intestine", RES MICROBIOL, vol. 158, 2007, pages 694 - 701
L. ECKMANN: "Animal Models of Inflammatory Bowel Disease: Lessons from Enteric Infections", ANN N Y ACAD SCI, vol. 1072, 2006, pages 28 - 38 8
L. M. HIGGINS ET AL.: "Citrobacter Rodentium Infection in Mice Elicits a Mucosal Thl Cytokine Response and Lesions Similar to Those in Murine Inflammatory Bowel Disease", INFECT IMMUN, vol. 67, 1999, pages 3031 - 9
L. T. AXELSSON ET AL.: "Production of a Broad Spectrum Antimicrobial Substance by Lactobacillus Reuteri", MICROBIAL ECOLOGY IN HEALTH AND DISEASE, 1989, pages 2
LAINE ET AL., EUR. J. BIOCHEM., vol. 103, no. 3, 1980, pages 447 - 481
LIN ET AL.: "Probiotic Lactobacillus Reuteri Suppress Proinflammatory Cytokines Via C-Jun", INFLAMM BOWEL DIS., vol. 14, 2008, pages 1068 - 83
LUPERCHIO; SCHAUER: "Molecular Pathogenesis of Citrobacter rodentium and Transmissible Murine Colonic Hyperplasia", MICROBES INFECT, vol. 3, 2001, pages 333 - 40
LUPERCHIO; SCHAUER: "Molecular Pathogenesis of Citrobacter rodentium and Transmissible Murine Colonic Hyperplasia", MICROBES INFECT., vol. 3, 2001, pages 333 - 40
LUPP, M. L. ET AL.: "Host-Mediated Inflammation Disrupts the Intestinal Microbiota and Promotes the Overgrowth of Enterobacteriaceae", CELL HOST MICROBE, vol. 2, 2007, pages 119 - 29
M. CARROLL ET AL.: "lterations in Composition and Diversity of the Intestinal Microbiota in Patients with Diarrhea-Predominant Irritable Bowel Syndrome", NEUROGASTROENTEROL MOTIL, vol. 24, 2012, pages 521 - 30
M. O. FREIRE, P. ET AL.: "Development of an Animal Model for Aggregatibacter Actinomycetemcomitans Biofilm-Mediated Oral Osteolytic Infection: A Preliminary Study", J PERIODONTOL, vol. 82, 2011, pages 778 - 89
M. T. BAILEY ET AL.: "Stressor Exposure Disrupts Commensal Microbial Populations in the Intestines and Leads to Increased Colonization by Citrobacter Rodentium", INFECT IMMUN, vol. 78, 2010, pages 1509 - 19
M. WLODARSKA ET AL.: "Antibiotic Treatment Alters the Colonic Mucus Layer and Predisposes the Host to Exacerbated Citrobacter Rodentium-Induced Colitis", INFECT IMMUN, vol. 79, 2011, pages 1536 - 45
MACHIELS ET AL.: "A Decrease of the Butyrate-Producing Species Roseburia hominis and Faecalibacterium prausnitzii Defines Dysbiosis in Patients with Ulcerative Colitis", GUT, 10 September 2013 (2013-09-10)
MACKOS ET AL., INFECTION AND IMMUNITY, vol. 81, no. 9, 2013, pages 3253 - 3262
MACKOS ET AL.: "Probiotic Lactobacillus reuteri Attenuates the Stressor-Enhanced Severity of Citrobacter Rodentium Infection", INFECT IMMUN, vol. 81, 2013, pages 3253 - 63
MACPHERSON ET AL., PCR 2: A PRACTICAL APPROACH, 1995
MACPHERSON ET AL.: "PCR 1: A Practical Approach", 1991, IRL PRESS AT OXFORD UNIVERSITY PRESS
MAUNDER ET AL.: "The role of stress in the development and clinical course of inflammatory bowel disease: epidemiological evidence", CURR MOL MED, vol. 8, 2008, pages 247 - 252
MESSOUDI ET AL.: "Beneficial psychological effects of a probiotic formulation (Lactobacillus helveticus R0052 and Bifidobacterium longum R0175) in healthy human volunteers", BR J NUTRI, vol. 105, 2011, pages 755 - 764
MORGAN ET AL.: "Dysfunction of the Intestinal Microbiome in Inflammatory Bowel Disease and Treatment", GENOME BIOL., vol. 13, 2012, pages R79
MUNDY, T. T. ET AL.: "Citrobacter rodentium of Mice and Man", CELL MICROBIOL, vol. 7, 2005, pages 1697 - 706
N. TAKEMURA ET AL.: "Inulin Prolongs Survival of Intragastrically Administered Lactobacillus Plantarum No. 14 in the Gut of Mice Fed a High-Fat Diet", J NUTR, vol. 140, 2010, pages 1963 - 9
NATARO; KAPER: "Diarrheagenic Escherichia Coli", CLIN MICROBIOL REV., vol. 11, 1998, pages 142 - 201
O. SCHREIBER ET AL.: "Lactobacillus Reuteri Prevents Colitis by Reducing P-Selectin-Associated Leukocyte- and Platelet-Endothelial Cell Interactions", AM J PHYSIOL GASTROINTEST LIVER PHYSIOL, vol. 296, 2009, pages G534 - 42
P. HEMARAJATA; J. VERSALOVIC: "Effects ofProbiotics on Gut Microbiota: Mechanisms of Intestinal Immunomodulation and Neuromodulation", THERAP ADV GASTROENTEROL, vol. 6, 2013, pages 39 - 51
PERBAL, A PRACTICAL GUIDE TO MOLECULAR CLONING, 1984
Q. XIA ET AL.: "Quantitative Analysis of Intestinal Bacterial Populations from Term Infants Fed Formula Supplemented with Fructo-Oligosaccharides", J PEDIATR GASTROENTEROL NUTR, vol. 55, 2012, pages 314 - 20
R. E. BLACK ET AL.: "Lancet", vol. 375, 2010, W. H. O. CHILD HEALTH EPIDEMIOLOGY REFERENCE GROUP OF, AND UNICEF, article "Global, Regional, and National Causes of Child Mortality in 2008: A Systematic Analysis", pages: 1969 - 87
R. MUNDY ET AL.: "Citrobacter Rodentium of Mice and Man", CELL MICROBIOL, vol. 7, 2005, pages 1697 - 706
S. A. LUPERCHIO; D. B. SCHAUER: "Molecular Pathogenesis of Citrobacter Rodentium and Transmissible Murine Colonic Hyperplasia", MICROBES INFECT, vol. 3, 2001, pages 333 - 40
S. D. GOODMAN, K. P. ET AL.: "Biofilms Can Be Dispersed by Focusing the Immune System on a Common Family of Bacterial Nucleoid-Associated Proteins", MUCOSAL IMMUNOL, vol. 4, 2011, pages 625 - 37
S. E. JONES; J. VERSALOVIC: "Probiotic Lactobacillus Reuteri Biofilms Produce Antimicrobial and Anti-Inflammatory Factors", BMC MICROBIOL, vol. 9, 2009, pages 35
S. FREDENBERG, M. ET AL.: "The Mechanisms of Drug Release in Poly(Lactic-Co-Glycolic Acid)-Based Drug Delivery Systems--a Review", INT J PHARM, vol. 415, 2011, pages 34 - 52
S. J. BEER ET AL.: "Poly (Lactic-Glycolic) Acid Copolymer Encapsulation of Recombinant Adenovirus Reduces Immunogenicity in Vivo", GENE THER, vol. 5, 1998, pages 740 - 6
S. L. LEBEIS ET AL.: "Protective and Destructive Innate Immune Responses to Enteropathogenic Escherichia Coli and Related a/E Pathogens", FUTURE MICROBIOL, vol. 3, 2008, pages 315 - 28
S. S. JUSTICE, B. ET AL.: "Aberrant Community Architecture and Attenuated Persistence of Uropathogenic Escherichia Coli in the Absence of Individual Ihf Subunits", PLOS ONE, vol. 7, 2012, pages E48349
SCHREIBER ET AL.: "Lactobacillus reuteri Prevents Colitis by Reducing P-Selectin-Associated Leukocyte- and Platelet-Endothelial Cell Interactions", AM J PHYSIOL GASTROINTEST LIVER PHYSIOL, vol. 296, 2009, pages G534 - 42
SINHA ET AL.: "Poly-Epsilon-Caprolactone Microspheres and Nanospheres: An Overview", INT J PHARM, vol. 278, 2004, pages 1 - 23
T. ABEE; O. P. KUIPERS: "Understanding Microbial Behavior within and Outside the Host to Improve Food Functionality and Safety", CURR OPIN BIOTECHNOL, vol. 22, 2011, pages 133 - 5
TOPUZ; O. OKAY: "Formation of Hydrogels by Simultaneous Denaturation and CrossLinking of DNA", BIOMACROMOLECULES, vol. 10, 2009, pages 2652 - 61
V. R. SINHA ET AL.: "Poly-Epsilon-Caprolactone Microspheres and Nanospheres: An Overview", INT J PHARM, vol. 278, 2004, pages 1 - 23
WALER ET AL.: "The Manitoba IBD cohort study: a population-based study of the prevalence of lifetime and 12-month anxiety and mood disorders", AM J GASTROENTEROL, vol. 103, 2008, pages 1989 - 1997
WLODARSKA, B. ET AL.: "Antibiotic Treatment Alters the Colonic Mucus Layer and Predisposes the Host to Exacerbated Citrobacter rodentium-Induced Colitis", INFECT IMMUN, vol. 79, 2011, pages 1536 - 45
WLODARSKA, B. ET AL.: "Antibiotic Treatment Alters the Colonic Mucus Layer and Predisposes the Host to Exacerbated Citrobacter rodentium-Induced Colitis", INFECT IMMUN., vol. 79, 2011, pages 1536 - 45
X. C. MORGAN ET AL.: "Dysfunction of the Intestinal Microbiome in Inflammatory Bowel Disease and Treatment", GENOME BIOL, vol. 13, 2012, pages R79
X. M. BEN ET AL.: "Low Level of Galacto-Oligosaccharide in Infant Formula Stimulates Growth of Intestinal Bifidobacteria and Lactobacilli", WORLD J GASTROENTEROL, vol. 14, 2008, pages 6564 - 8
Y. P. LIN ET AL.: "Probiotic Lactobacillus Reuteri Suppress Proinflammatory Cytokines Via C-Jun", INFLAMM BOWEL DIS, vol. 14, 2008, pages 1068 - 83

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